KR100990266B1 - Apoptosis sensitivity to apo2l/trail by testing for galnac-t14 expression in cells/tissues - Google Patents

Abstract

Methods and assays examining expression of one or more biomarkers in a mammalian tissue or cell sample are provided. According to the disclosed methods and assays, detection of the expression of GalNac-T related molecules, such as GalNac-T14 or GalNac-T3, is predictive or indicative that the tissue or cell sample will be sensitive to apoptosis-including agents such as Apo2L/TRAIL and anti-DR5 agonist antibodies. Kits and articles of manufacture are also provided.

Description

Apoptosis sensitivity to APO2L / TRAIL predicted by testing for BALC-T14 expression in cells / tissues {APOPTOSIS SENSITIVITY TO APO2L / TRAIL BY TESTING FOR GALNAC-T14 EXPRESSION IN CELLS / TISSUES}

Related application

This application claims priority to US patent provisional application 60 / 708,677 (filed Aug. 16, 2005) and US patent provisional application 60 / 808,076 (filed May 24, 2006), the contents of which are hereby incorporated by reference. do.

The invention described herein relates to methods and assays for detecting biomarkers that predict the sensitivity of mammalian cells to Apo2L / TRAIL and / or death receptor agonist antibodies. More particularly, the present invention relates to methods and assays for detecting molecules associated with proteins of the GalNac-T family that predict the sensitivity of mammalian cancer cells to Apo2L / TRAIL or death receptor agonist antibodies, such as DR4 or DR5 agonist antibodies. It is about.

Various ligands and receptors belonging to the tumor necrosis factor (TNF) superfamily have been identified in the art. Such ligands include tumor necrosis factor-alpha ("TNF-alpha"), tumor necrosis factor-beta ("TNF-beta" or "lymphotoxin-alpha"), lymphotoxin-beta ("LT-beta"), and CD30 ligands. , CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, LIGHT, Apo-1 ligand (also referred to as Fas ligand or CD95 ligand), Apo-2 ligand (also referred to as Apo2L or TRAIL), Apo -3 ligand (also referred to as TWEAK), APRIL, OPG ligand (also referred to as RANK ligand, ODF, or TRANCE), and TALL-1 (also referred to as BlyS, BAFF or THANK) For example, Ashkenazi, Nature Review, 2: 420-430 (2002); Ashkenazi and Dixit, Science, 281: 1305-1308 (1998); Ashkenazi and Dixit, Curr. Opin. Cell Biol., 11: 255-260 (2000); Golstein, Curr. Biol., 7: 750-753 (1997); Wallach, Cytokine Reference, Academic Press, 2000, pages 377-411; Locksley et al., Cell 104: 487-501 (2001); Gruss and Dower, Blood, 85: 3378-3404 (1995); Schmid et al. Proc. Natl. Acad. Sci., 83: 1881 (1986); Dealtry et al., Eur. J. Immunol., 17: 689 (1987); Pitti et al., J. Biol. Chem 271: 12687-12690 (1996); Wiley et al., Immunity, 3: 673-682 (1995); Browning et al., Cell, 72: 847-856 (1993); Armitage et al. Nature, 357: 80-82 (1992); WO 97/01633, published January 16, 1997; WO 97/25428 (published 17 July 1997); Marsters et al., Curr. Biol., 8: 525-528 (1998); Chicheportiche et al., Biol. Chem., 272: 32401-32410 (1997); Hahne et al., J. Exp. Med., 188: 1185-1190 (1998); WO 98/28426 published July 2, 1998; WO 98/46751 published October 22, 1998; WO / 98/18921, published May 7, 1998; Moore et al., Science, 285: 260-263 (1999); Shu et al., J. Leukocyte Biol., 65: 680 (1999); Schneider et al., J. Exp. Med., 189: 1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem., 274: 15978-15981 (1999).

Induction of various cellular responses mediated by such TNF family ligands is typically initiated by their binding to specific cellular receptors. Some, but not all, TNF family ligands bind to cell surface "kill receptors" through which they induce a variety of biological activities, activating enzymes that perform caspases or apoptosis or apoptosis pathways (Salvesen et. al., Cell, 91: 443-446 (1997)]. Members of the TNF receptor superfamily identified to date include TNFR1, TNFR2, TACI, GITR, CD27, OX-40, CD30, CD40, HVEM, Fas (also referred to as Apo-1 or CD95), DR4 (TRAIL-R1) Also referred to), DR5 (also referred to as Apo-2 or TRAIL-R2), DcR1, DcR2, osteoprotegerin (OPG), RANK and Apo-3 (also referred to as DR3 or TRAMP) See, eg, Ashkenazi, Nature Reviews, 2: 420-430 (2002); Ashkenazi and Dixit, Science, 281: 1305-1308 (1998); Ashkenazi and Dixit, Curr. Opin. Cell Biol., 11: 255-260 (2000); Golstein, Curr. Biol., 7: 750-753 (1997); Wallach, Cytokine Reference, Academic Press, 2000, pages 377-411; Locksley et al. , Cell, 104: 487-501 (2001); Gross and Dower, Blood, 85: 3378-3404 (1995); Hohman et al., J. Biol. Chem., 264: 14927-14934 (1989 Brockhaus et al., Proc. Natl. Acad. Sci., 87: 3127-3131 (1990); EP 417,563, published March 20, 1991; Loetscher et al., Cell, 61: 351 (1990); Schall et al., Cell, 61: 361 (19 Smith et al., Science, 248: 1019-1023 (1990); Lewis et al., Proc. Natl. Acad. Sci., 88: 2830-2834 (1991); Goodwin et al., Mol. Cell. Biol., 11: 3020-3026 (1991); Stamenkovic et al., EMBO J., 8: 1403-1410 (1989); Mallett et al., EMBO J., 9: 1063-1068 (1990); Anderson et al., Nature, 390: 175-179 (1997); Chicheportiche et al., J. Biol. Chem., 272: 32401-32410 (1997); Pan et al., Science, 276: 111-113 (1997); Pan et al., Science, 277: 815-818 (1997); Sheridan et al., Science, 277: 818-821 (1997); Degli-Esposti et al., J. Exp. Med., 186: 1165-1170 (1997); Marsters et al., Curr. Biol., 7: 1003-1006 (1997); Tsuda et al., BBRC, 234: 137-142 (1997); Nocentini et al., Proc. Natl. Acad. Sci., 94: 6216-6221 (1997); (von Bulow et al., Science, 278: 138-141 (1997)).

Most of these TNF receptor family members share the typical structure of cell surface receptors, including extracellular regions, transmembrane regions and intracellular regions, while others are soluble proteins that are free of transmembrane and intracellular domains in nature. Is found. The extracellular portion of a typical TNFR contains a repeating amino acid sequence pattern of multiple cysteine-rich domains (CRDs) starting at the NH ₂ -terminus.

Ligands called Apo-2L or TRAIL have been identified many years ago as members of the TNF family of cytokines (see, eg, Wiley et al., Immunity, 3: 673-682 (1995); Pitti et al. , J. Biol. Chem., 271: 12697-12690 (1996); WO 97/01633; WO 97/25428; US Patent 5,763,223 issued June 9, 1998; US Patent 6,284,236 (September 4, 2001) See work permit). Full length native sequence human Apo2L / TRAIL polypeptide is a 281 amino acid long type II transmembrane protein. Some cells may produce naturally soluble forms of the polypeptide through enzymatic cleavage of the extracellular region of the polypeptide (Mariani et al., J. Cell. Biol., 137: 221-229 (1997)). . Crystallographic studies of soluble forms of Apo2L / TRAIL have shown homotrimeric structures similar to those of TNF and other related proteins (Hymowitz et al., Molec. Cell, 4: 563-571 (1999); Cha et al. al., Immunity, 11: 253-261 (1999); Mongkolsapaya et al., Nature Structural Biology, 6: 1048 (1999); Hymowitz et al., Biochemistry, 39: 633-644 (2000); ). However, unlike other TNF family members, Apo2L / TRAIL has three cysteine residues (position 230 of each subunit in the homotrimer) coordinating with the zinc atom, and zinc binding is important for trimer stability and biological activity. It was found to have unique structural features in that sense (Hymowitz et al., Supra; Bodmer et al., J. Biol. Chem., 275: 20632-20637 (2000)).

It has been reported in the literature that Apo2L / TRAIL may play a role in immune system modulation including autoimmune diseases such as rheumatoid arthritis (see, eg, Thomas et al., J. Immunol., 161: 2195-2200). (1998)]; Johnsen et al., Cytokine, 11: 664-672 (1999); Griffith et al., J. Exp. Med., 189: 1343-1353 (1999); Song et al. , J. Exp. Med., 191: 1095-1103 (2000).

Soluble forms of Apo2L / TRAIL have also been reported to induce apoptosis in colon, lung, breast, prostate, bladder, kidney, ovarian and brain tumors, as well as various cancer cells including melanoma, leukemia, and multiple myeloma. (E.g., Wiley et al., Supra; Pitti et al., Supra); US Pat. No. 6,030,945, issued February 29, 2000; US Pat. No. 6,746,668, issued June 8, 2004. Riger et al., FEBS Letters, 427: 124-128 (1998); Ashkenazi et al., J. Clin. Invest., 104: 155-162 (1999); Walzakzak et al., Nature Med., 5: 157-163 (1999); Keane et al., Cancer Research, 59: 734-741 (1999); Mizutani et al., Clin. Cancer Res., 5: 2605-2612 (1999); Gazitt, Leukemia, 13: 1817-1824 (1999); Yu et al., Cancer Res., 60: 2384-2389 (2000); Chinnaiyan et al., Proc. Natl. Acad. Sci., 97: 1754-1759 (2000). In vivo studies in a murine tumor model further suggested that Apo2L / TRAIL can exert substantial anti-tumor effects alone or in combination with chemotherapy or radiotherapy (eg, Ashkenazi et al. Walzcak et al., Supra; Gliniak et al., Cancer Res., 59: 6153-6158 (1999); Chinnaiyan et al., Supra; Roth et al. , Biochem. Biophys. Res. Comm., 265: 1999 (1999); see PCT Application US / 00/15512; PCT Application US / 01/23691. Unlike many types of cancer cells, most normal human cell types appear to be resistant to induction of apoptosis by specific recombinant forms of Apo2L / TRAIL (Ashkenazi et al., Supra); Walzcak et al., Supra literature]). Jo et al. Reported that soluble form of Apo2L / TRAIL with polyhistidine-tag induced apoptosis in normal isolated human hepatocytes in vitro but not in non-human hepatocytes ( Jo et al., Nature Med., 6: 564-567 (2000); see also Nagata, Nature Med., 6: 502-503 (2000). For example, depending on the presence or absence of tag molecules, zinc content, and trimer content percent, it is believed that certain recombinant Apo2L / TRAIL preparations may vary in terms of biochemical properties and biological activity for diseased cells versus normal cells. (Lawrence et al., Nature Med., Letter to the Editor, 7: 383-385 (2001); Qin et al., Nature Med., Letter to the Editor, 7: 385-386 (2001)) Reference).

Apo2L / TRAIL has been found to bind to at least five different receptors. At least two of the receptors that bind Apo2L / TRAIL include a functional cytoplasmic death domain. One such receptor was termed “DR4” (alternatively referred to as TR4 or TRAIL-R1) (Pan et al., Science, 276: 111-113 (1997)); WO 98/32856 (7 1998) Published 30 March); WO 99/37684 published 29 July 1999; WO 00/73349 published 7 December 2000; US 2003/0036168 published 20 February 2003; US 6,433,147 (August 13, 2002); see US 6,461,823 (October 8, 2002) and US 6,342,383 (January 29, 2002).

Another such receptor for Apo2L / TRAIL has been referred to as DR5 (alternatively Apo-2; also referred to as TRAIL-R or TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER) (eg Sheridan et al., Science, 277: 818-821 (1997); Pan et al., Science, 277: 815-818 (1997); WO 98/51793, published November 19, 1998. WO 98/41629 published September 24, 1998; Screaton et al., Curr. Biol., 7: 693-696 (1997); Walczak et al., EMBO J., 16: 5386-. 5387 (1997); Wu et al., Nature Genetics, 17: 141-143 (1997); WO 98/35986, published August 20, 1998; EP 870,827, published October 14, 1998 ; WO 98/46643 published October 22, 1998; WO 99/02653 published January 21, 1999; WO 99/09165 published February 25, 1999; WO 99/11791, 1999 Published March 11); WO 03/042367 published May 22, 2003; WO 02/097033 published December 5, 2002; WO 03/038043 published May 8, 2003; US 2002/0072091 (released August 13, 2002); US 2002/0098550 (released December 7, 2001); US 6,313,269 (issued 6 December 2001); US 2001/0010924 (released 2 August 2001); US 2003/01255540 (released 3 July 2003); US 2002/0160446 (Oct 31, 2002) Published); US 2002/0048785 (published April 25, 2002); US 2004/0141952 (published July 22, 2004); US 2005/0129699 (published June 16, 2005); US 2005/0129616 published June 16, 2005; US 6,342,369 (February 2002); US 6,569,642 issued May 27, 2003; US 6,072,047 issued June 6, 2000; US 6,642,358 issued November 4, 2003; US 6,743,625 issued June 1, 2004). Like DR4, it has been reported that DR5 can carry a signal of apoptosis when it contains a cytoplasmic death domain and the ligand binds (or a molecule such as an agonist antibody that mimics the activity of the ligand). The crystal structure of the complex formed between Apo-2L / TRAIL and DR5 is described in Hymowitz et al., Molecular Cell., 4: 563-571 (1999).

When the ligand binds, both DR4 and DR5 can independently trigger apoptosis by recruiting and activating caspase-8, an apoptosis initiator, via a death domain containing adapter molecule called FADD / Mort1 (Kischkel et al. al., Immunity, 12: 611-620 (2000); Sprick et al., Immunity, 12: 599-609 (2000); Bodmer et al., Nature Cell Biol., 2: 241-243 ( 2000)]).

Apo2L / TRAIL has also been reported to bind to receptors called DcR1, DcR2 and OPG, which are believed to function as inhibitors rather than transducers of signaling (eg, DCR1 (TRID, LIT or TRAIL-R3). (Also referred to as Pan et al., Science, 276: 111-113 (1997)); Sheridan et al., Science, 277: 818-821 (1997); McFarlane et al., J Biol. Chem., 272: 25417-25420 (1997); Schneider et al., FBBS Letters, 416: 329-334 (1997); Degli-Esposti et al., J. Exp. Med., 186: 1165-1170 (1997); and Mongkolsapaya et al., J. Immunol., 160: 3-6 (1998); for DCR2 (also referred to as TRUNDD or TRAIL-R4) [Marsters et al. Curr. Biol., 7: 1003-1006 (1997); Pan et al., FEBS Letters, 424: 41-45 (1998); Degli-Esposti et al., Immunity, 7: 813- 820 (1997), for SIMG, see Simonet et al., Supra. Unlike DR4 and DR5, DcR1 and DcR2 receptors do not carry apoptosis signals.

Certain antibodies that bind to DR4 and / or DR5 receptors have been reported in the literature. For example, anti-DR4 antibodies directed against the DR4 receptor and having agonist or apoptotic activity in certain mammalian cells are described, for example, in WO 99/37684 (July 29, 1999); WO 00/73349 published July 12, 2000; WO 03/066661, published August 14, 2003. See also, Griffith et al., J. Immunol., 162: 2597-2605 (1999); Chuntharapai et al., J. Immunol., 166: 4891-4898 (2001); WO 02/097033 published December 2, 2002; WO 03/042367 published May 22, 2003; WO 03/038043, published May 8, 2003; WO 03/037913 published May 8, 2003; US 2003/0073187 (published April 17, 2003); See US 2003/0108516, published June 12, 2003. Certain anti-DR5 antibodies are likewise described, for example WO 98/51793 (published November 8, 1998); Griffith et al., J. Immunol., 162: 2597-2605 (1999); Ichikawa et al., Nature Med., 7: 954-960 (2001); Hylander et al., "An Antibody to DR5 (TRAIL-Receptor 2) Suppresses the Growth of Patient Derived Gastrointestinal Tumors Grown in SCID mice", Abstract, 2d International Congress on Monoclonal Antibodies in Cancers, Aug. 29-Sept. 1, 2002, Banff, Alberta, Canada; WO 03/038043, published May 8, 2003; WO 03/037913 published May 8, 2003; See US 2003/0180296, published September 25, 2003. In addition, certain antibodies with crossreactivity to both DR4 and DR5 receptors have been described (see, eg, US Pat. No. 6,252,050, issued June 26, 2001).

Summary of the Invention

The invention disclosed herein provides methods and assays for investigating the expression of one or more biomarkers in a mammalian tissue or cell sample, wherein expression of the one or more such biomarkers is such that Apo2L / TRAIL or anti- Predict whether to be sensitive to agents such as DR5 agonist antibodies. In various embodiments of the invention, the methods and assays examine the expression of molecules of the GalNac-T family of proteins, in particular GalNAc-T14 or GalNAc-T3.

As discussed above, most normal human cell types appear to be resistant to induction of apoptosis by specific recombinant forms of Apo2L / TRAIL (Ashkenazi et al., Supra); Walzcak et al., Supra ]). It has also been observed that some populations of human disease cell types (such as certain populations of cancer cells) are resistant to induction of apoptosis by certain recombinant forms of Apo2L / TRAIL (Ashkenazi et al., J. Clin. Invest., 1999, supra, Walczak et al., Nature Med., 1999, supra. As a result, by investigating mammalian tissue or cell samples by assay for expression of selected biomarkers, it is possible to conveniently and efficiently obtain information useful for evaluating suitable or effective therapies for treating patients. For example, information obtained from assays that detect GalNac-T14 expression in mammalian tissue or cell samples may be used to determine optimal treatment for patients suffering from disorders such as cancer or immune-related diseases, such as auto-immune disorders. Useful data that can be used to determine Apo2L / TRAIL or death receptor agonist antibodies) can be provided to the physician.

The present invention provides a method for predicting the sensitivity of a mammalian tissue or cell sample (such as cancer cells) to Apo2L / TRAIL or death receptor agonist antibodies. In certain embodiments, the method comprises obtaining a mammalian tissue or cell sample and examining the tissue or cell for expression of GalNac-T14. The methods can be performed in a variety of assay formats, including assays for detecting mRNA and / or protein expression, enzyme activity assays, and other assays discussed herein. Determination of the expression of GalNac-T14 in such tissues or cells will predict that such tissues or cells will be sensitive to apoptosis-inducing activity of Apo2L / TRAIL and / or death receptor antibodies. In optimal embodiments, tissues or cells may also be examined for expression of DR4, DR5, DcR1 or DcR2 receptors.

An additional embodiment of the present invention is directed to obtaining a mammalian tissue or cell sample, examining the tissue or cell for expression of GalNac-T14, and when it is determined that the tissue or cell sample expresses GalNac-T14. And inducing apoptosis in mammalian tissue or cell sample, comprising exposing the tissue or cell sample to an effective amount of Apo2L / TRAIL or a death receptor agonist antibody. Examining the expression of GalNac-T14 in the method can be performed in a variety of assay formats, including assays for detecting mRNA and / or protein expression, enzyme activity assays, and other assays discussed herein. In an optimal embodiment, the method also includes examining the tissue or cell sample for expression of the DR4, DR5, DcR1 or DcR2 receptor. Optionally, the tissue or cell sample comprises cancerous tissue or cells. Optionally, the tissue or cell sample comprises non-small cell lung cancer cells, pancreatic cancer cells, breast cancer cells, or non-hodgkin lymphoma cells.

Additional methods of the invention include obtaining a tissue or cell sample from a mammal, examining the tissue or cell for expression of GalNac-T14, and when it is determined that the tissue or cell sample expresses GalNac-T14. A method of treating a mammalian disorder, such as an immune related disorder or cancer, comprising administering to the mammal an effective amount of an Apo2L / TRAIL or a death receptor agonist antibody. Investigating the expression of one or more biomarkers of the methods can be performed in a variety of assay formats, including assays for detecting mRNA and / or protein expression, enzyme activity assays, and other assays discussed herein. In an optimal embodiment, the method also includes examining the tissue or cell sample for expression of the DR4, DR5, DcR1 or DcR2 receptor. Optionally, the tissue or cell sample comprises cancer tissue or cells. Optionally, the method includes treating the cancer in a mammal. Optionally, in addition to administering an effective amount of Apo2L / TRAIL and / or death receptor agonist antibody, the method includes administering a chemotherapeutic agent (s) or radiation therapy to the mammal.

In a further embodiment of the invention, the aforementioned methods may comprise examining mammalian tissue or cells for expression of another GalNac-T molecule, such as GalNac-T3.

Further embodiments are described for example in the following claims:

1. obtaining a mammalian tissue or cell sample;

Investigating tissue or cell samples to detect expression of GalNac-T14

Wherein the expression of GalNac-T14 predicts that the tissue or cell sample is sensitive to the apoptosis-inducing activity of Apo2L / TRAIL, wherein the mammalian tissue or cell sample is sensitive to Apo2L / TRAIL. How to predict.

2. The method of paragraph 1, wherein said GalNac-T14 expression is investigated by detecting the expression of GalNac-T14 mRNA.

3. The method of paragraph 1, wherein said GalNac-T14 expression is investigated by immunohistochemistry.

4. The method of paragraph 1, further comprising the step of examining expression of DR4, DR5, DcR1 or DcR2 receptor in said tissue or cell sample.

5. The method of paragraph 1, wherein the tissue or cell sample comprises cancer tissue or cells.

6. The method of claim 5, wherein the cancer cell is pancreatic cancer, lymphoma, or non-small cell lung cancer cell or tissue.

7. obtaining a mammalian tissue or cell sample;

Examining a tissue or cell sample to detect expression of GalNac-T14, and

Following detection of the expression of GalNac-T14, exposing the tissue or cell sample to an effective amount of Apo2L / TRAIL

Including, a method of inducing apoptosis in mammalian tissue or cell sample.

8. The method of claim 7, wherein said GalNac-T14 expression is examined by testing for expression of GalNac-T14 mRNA.

9. The method of claim 7, wherein said GalNac-T14 expression is investigated by immunohistochemistry.

10. The method of claim 7, further comprising the step of examining expression of DR4, DR5, DcR1 or DcR2 receptor in said tissue or cell sample.

11. The method of claim 7, wherein said tissue or cell sample comprises cancer tissue or cells.

12. The method of paragraph 11, wherein the cancer cell is pancreatic cancer, lymphoma, or non-small cell lung cancer cell or tissue.

13. The method of claim 7, wherein said cells are exposed to an effective amount of Apo2L / TRAIL polypeptide comprising amino acids 114-281 of FIG.

14. obtaining a tissue or cell sample from the mammal;

Examining a tissue or cell sample to detect expression of GalNac-T14, and

Following detection of the expression of GalNac-T14, administering an effective amount of Apo2L / TRAIL to the mammal

A method of treating a mammalian disorder, such as an immune related disorder or cancer, comprising.

15. The method of paragraph 14, wherein said GalNac-T14 expression is investigated by detecting the expression of GalNac-T14 mRNA.

16. The method of paragraph 14, wherein said GalNac-T14 expression is investigated by immunohistochemistry.

17. The method of claim 14, further comprising examining the expression of DR4, DR5, DcR1 or DcR2 receptors in said tissue or cell.

18. The method of paragraph 14, wherein the tissue or cell sample comprises cancer tissue or cells.

19. The method of paragraph 18, wherein the cancer cell or tissue comprises pancreatic cancer, lymphoma or non-small cell lung cancer cells or tissue.

20. The method of paragraph 14, wherein the mammal is administered an effective amount of Apo2L / TRAIL polypeptide comprising amino acids 114-281 of FIG. 1.

21. The method of paragraph 14, wherein the mammal is also administered a chemotherapeutic agent (s) or radiation therapy.

22. The method of paragraph 14, wherein the mammal is also administered a cytokine, cytotoxic agent, or growth inhibitory agent.

23. The method of claim 7, wherein said Apo2L / TRAIL polypeptide is linked to a polyethylene glycol molecule.

24. The method of claim 14, wherein said Apo2L / TRAIL polypeptide is linked to a polyethylene glycol molecule.

25. obtaining a mammalian tissue or cell sample;

Investigating tissue or cell samples to detect expression of GalNac-T14

Wherein the expression of GalNac-T14 predicts that the tissue or cell sample is sensitive to apoptotic-induced activity of the death receptor antibody, the sensitivity of the mammalian tissue or cell sample to the death receptor antibody. How to predict.

26. The method of paragraph 25, wherein said GalNac-T14 expression is investigated by detecting the expression of GalNac-T14 mRNA.

27. The method of paragraph 25, wherein said GalNac-T14 expression is investigated by immunohistochemistry.

28. The method of paragraph 25, further comprising examining the expression of DR4, DR5, DcR1 or DcR2 receptor in said tissue or cell sample.

29. The method of paragraph 25, wherein the tissue or cell sample comprises cancer tissue or cells.

30. The method of claim 29, wherein the cancer cell is pancreatic cancer, lymphoma or non-small cell lung cancer cell or tissue.

31. The method of paragraph 25, wherein said death receptor antibody is an agonist anti-DR4 or anti-DR5 antibody.

32. obtaining a mammalian tissue or cell sample;

Examining a tissue or cell sample to detect expression of GalNac-T14, and

Following detection of the expression of GalNac-T14, exposing the tissue or cell sample to an effective amount of a death receptor antibody

Including, a method of inducing apoptosis in mammalian tissue or cell sample.

33. The method of paragraph 32, wherein said GalNac-T14 expression is examined by testing for expression of GalNac-T14 mRNA.

34. The method of claim 32, wherein said GalNac-T14 expression is investigated by immunohistochemistry.

35. The method of claim 32, further comprising examining the expression of DR4, DR5, DcR1 or DcR2 receptors in said tissue or cell sample.

36. The method of claim 32, wherein the tissue or cell sample comprises cancer tissue or cells.

37. The method of paragraph 36, wherein the cancer cell is pancreatic cancer, lymphoma or non-small cell lung cancer cell or tissue.

38. The method of claim 32, wherein said cells are exposed to an effective amount of agonist DR4 or DR5 antibody.

39. The method of claim 38, wherein said cells are exposed to an effective amount of agonist DR5 antibody that binds to the DR5 receptor shown in FIG. 3A.

40. obtaining a tissue or cell sample from a mammal;

Examining a tissue or cell sample to detect expression of GalNac-T14, and

Following detection of the expression of GalNac-T14, administering to said mammal an effective amount of a death receptor antibody

A method of treating a mammalian disorder, such as an immune related disorder or cancer, comprising.

41. The method of paragraph 40, wherein said GalNac-T14 expression is investigated by detecting the expression of GalNac-T14 mRNA.

42. The method of paragraph 40, wherein said GalNac-T14 expression is investigated by immunohistochemistry.

43. The method of claim 40, further comprising examining the expression of DR4, DR5, DcR1 or DcR2 receptor in said tissue or cell.

44. The method of claim 40, wherein the tissue or cell sample comprises cancer tissue or cells.

45. The method of 44, wherein the cancer cells or tissues comprise pancreatic cancer, lymphoma or non-small cell lung cancer cells or tissues.

46. The method of paragraph 40, wherein the mammal is administered an effective amount of an anti-DR4 or DR5 antibody.

47. The method of paragraph 40, wherein the mammal is also administered chemotherapeutic agent (s) or radiation therapy.

48. The method of paragraph 40, wherein the mammal is also administered a cytokine, cytotoxic agent, or growth inhibitory agent.

1 shows the nucleotide sequence of human Apo-2 ligand cDNA (SEQ ID NO: 2) and its derived amino acid sequence (SEQ ID NO: 1). "N" at nucleotide position 447 is used to indicate that the nucleotide base may be "T" or "G".

2A and 2B show the nucleotide sequence of cDNA (SEQ ID NO: 4) and its derived amino acid sequence (SEQ ID NO: 3) for full length human DR4. Respective nucleotide and amino acid sequences for human DR4 are also reported in Pan et al., Science, 276: 111 (1997).

3A shows the 411 amino acid sequence (SEQ ID NO: 5) of human DR5 as disclosed in WO 98/51793 on November 19, 1998. FIG. Transcription splice variants of human DR5 are known in the art. These DR5 splice variants encode the 440 amino acid sequences (SEQ ID NO: 6) of human DR5 shown in FIGS. 3B and 3C, as published in WO 98/35986 on August 20, 1998.

3D1 to 3D3 show the nucleotide sequence of cDNA (SEQ ID NO: 7) and its derived amino acid sequence (SEQ ID NO: 8) for full length human DcR1. Respective nucleotide and amino acid sequences for human DcR1 (and specific domains thereof) are also shown and described in WO 98/58062.

3E1 and 3E2 show the nucleotide sequence of cDNA (SEQ ID NO: 9) and its derived amino acid sequence (SEQ ID NO: 10) for full length human DcR2. Respective nucleotide and amino acid sequences for human DcR2 (and specific domains thereof) are also shown in WO 99/10484.

4A1-4A4 show the nucleotide sequence of human GalNac-T14 (SEQ ID NO: 11) and its derived amino acid sequence (SEQ ID NO: 12). Such sequences are also described in Wang et al., BBRC, 300: 738-744 (2003).

4B1-4B4 show the nucleotide sequence of human GalNac-T3 (SEQ ID NO: 13) and its derived amino acid sequence (SEQ ID NO: 14). Such sequences are described in Bennett et al., J. Biol. Chem., 271: 17006-17012 (1996).

5 shows Apo2L (+ 0.5% fetal bovine serum “FBS” or 10% FBS) or crosslinked “XL” or uncrosslinked DR5 monoclonal antibody “DR5 ab, as measured in the MTT cytotoxicity assay. IC50 Summary Plot of Data Obtained from Analyzing Non-Small Cell Lung Cancer (“NSCLC”) Cell Lines for Sensitivity or Resistance to Apoptotic Activity of “(+ 0.5% Fetal Bovine Serum“ FBS ”or 10% FBS) to provide.

6 shows Apo2L (+ 0.5% fetal bovine serum “FBS” or 10% FBS) or crosslinked “XL” or uncrosslinked DR5 monoclonal antibody “DR5 ab” (+ as measured in the MTT cytotoxicity assay). An IC50 summary chart of the data obtained from analyzing pancreatic cancer cell lines for sensitivity or resistance to apoptotic activity of 0.5% fetal bovine serum "FBS" or 10% FBS) is provided.

FIG. 7 shows Apo2L (+ 10% fetal bovine serum “FBS”) or crosslinked “XL” or uncrosslinked DR5 monoclonal antibody “DR5 ab” (+ 10% fetal calf) as measured in MTT cytotoxicity assay. IC50 Summary Plots of data obtained from analyzing non-Hodgkin's lymphoma cancer (“NHL”) cell lines for sensitivity or resistance to apoptotic activity of serum “FBS”) are provided.

8A and 8B are comparisons of sensitivity (“sen”) or resistance (“RES”) of selected NSCLC, pancreatic cancer, and NHL cell lines to DR5 antibodies, as measured by GalNac-T14 mRNA expression, and GalNac-T14 Provides a correlation for the expression of.

9A and 9B provide bar chart graphs of various NSCLC, pancreatic cancer, and NHL cell lines ranked in descending order by level of GalNac-T14 mRNA expression pattern.

10A-D illustrate the differential expression of specific O-glycosylation enzymes in Apo2L / TRAIL-sensitive and -resistant cancer cell lines: (A) Cell viability after incubation with various doses of Apo2L / TRAIL. IC50s for each cell line were computerized as concentrations of Apo2L / TRAIL providing a 50% loss of viability. Each cell viability experiment was repeated three or more times in the presence of low (0.5%) and high (10%) fetal bovine serum. Black, gray, or white symbols represent cell lines that are highly sensitive to Apo2L / TRAIL, moderately sensitive or resistant cell lines, respectively. (B) ppGalNAcT-14 mRNA expression levels in pancreas and malignant melanoma cell lines (probe set 219271_at). Cell lines are sorted by tissue type and sensitivity to Apo2L / TRAIL. Black, gray, or white bars depict cell lines as in A. (C) mRNA expression levels of Fut-6 (top panel, probe set 211885_x_at) and ppGalNAcT-3 (bottom panel, probe set 203397_s_at) in colorectal cancer cell lines. Cell lines are aligned as in B. P values in panels B and C are based on Fisher's validation of the correlation between cell expression (including high and moderate) and mRNA expression above cutoff. (D) Effect of Apo2L / TRAIL on the growth of established tumor xenografts. Vehicle or Apo2L / TRAIL (60 mg / kg / day) in athymic nude mice with GalNAcT-3 / Fut-6-positive (left panel) or GalNAcT-3 / Fut-6-negative (right panel) tumors , Intraperitoneally on days 0-4) and tumor volume was monitored (mean + SE, N = 10 mice / group).

11 illustrates that the modulation of certain O-glycosylation enzymes changes the sensitivity to Apo2L / TRAIL. (A) Colo205 cells were preincubated with pan O-glycosylase inhibitor benzyl-GalNAc (bGalNAc), treated with Apo2L / TRAIL for 24 hours, and cell viability was determined (DMSO = vehicle control). (B) PSN-1 (pancreatic carcinoma) and Hs294T (melanoma) cells were transfected with caspase-8 or ppGalNAcT-14 siRNA for 48 hours, incubated with Apo2L / TRAIL for 24 hours, and cell viability was determined It was. SiRNA duplex (Dharmacon) for non-targeting sequences was used as a control (NTC). (C) DLD-1 colorectal carcinoma cells were transfected with siRNA with ppGalNAcT-3 or Fut-6 and tested as in B. (D) HEK293 cells were co-transfected with plasmids encoding the indicated genes together with ppGalNAcT-14 or the vector control. Apoptosis was measured by Annexin V staining at 24 hours (left panel). H1569 melanoma cells were transduced with retrovirus or control retrovirus indicating ppGalNAcT-14 expression; The resulting cell line pools were treated with Apo2L / TRAIL for 24 hours and cell viability was determined (right panel). Western blot analysis using anti-FLAG antibodies was used to demonstrate expression of epitope-tagged ppGalNAcT-14.

12 (A) illustrates the analysis of caspase cascade induced by Apo2L / TRAIL. PSN-1 and DLD-1 cells were transfected for 48 hours with siRNA for ppGalNAcT-14 or Fut-6, respectively. Cells were treated with Apo2L / TRAIL for 4 or 8 hours and cell lysates were subjected to immunoblot with antibodies specific for caspase-8, Bid, caspase-9, caspase-3, or actin as a loading control. Analyzed by. (B) PSN-1 cells were transfected with ppGalNAcT-14 siRNA as in A, treated with Apo2L / TRAIL for 4 hours, and caspase-3 / 7 enzyme activity in cell lysates was determined. (C) Analysis of Apo2L / TRAIL DISC. PSN-1 cells were transfected with ppGalNAcT-14 siRNA as in A. FLAG-Apo2L / TRAIL (1 mg / ml) was added for 0-60 minutes, cells were lysed and immunoprecipitated with anti-FLAG antibody. DISC-associated FADD, caspase-8, DR4 were detected by immunoblot. (D) PSN-1 cells were transfected, treated and DISC immunoprecipitated as in C, and DISC-associated caspase-8 enzyme activity was measured as previously described (Sharp et al., J. Biol). Chem., 280: 19401 (2005)].

FIG. 13 illustrates monosaccharide analysis of recombinant human DR5 (long splice variants) produced in CHO cells, performed by (A) HPAEC-PAD (high performance anion-exchange chromatography-pulse amperometric detection). (B) Human Apo2L / TRAIL receptor (long amino acid 440 form "hDR5L" of human DR5, short amino acid 411 form "hDR5S" and hDR4 of human DR5), murine DR5 (mDR5), human Fas (hFas) and human TNFR1 Sequence comparison of (hTNFR1). Boxes indicate putative O-glycosylation sites. (C) Immunoblot analysis of whole cell lysates corresponding to D. DR5L-5T and DR5S-5T are constructs containing 5 threonine → alanine substitutions in residues that are potential O-glycosylation sites, while DR5L-5T3S and DR5S-5T3S are 5 threonine → alanine and 3 serine → alanine substitutions It is a construct containing. (D) HEK293 cells were co-transfected with the indicated DR5 construct with the vector or ppGalNAcT-14 plasmid for 48 hours and apoptosis was measured by Annexin V staining. (E) skin (SCC = squamous cell carcinoma), lung, pancreas (Panc), breast, ovary (Ov), endometrium (Endo), bladder (Bla, TCC = transitional cell carcinoma) and NHL (FL = follicular lymphoma) , MRNA expression level for ppGalNAcT-14 in primary human tumor samples from cancer of DLBCL = diffuse large B-cell lymphoma) (Affymetrix chip, probe set 219271_at). Median expression of the samples is indicated by gray horizontal bars for each class. Cutoff values of 500 and 200 (melanoma) corresponding to cell line data from FIG. 10B are indicated.

FIG. 14 (A) illustrates reduced mRNA expression of ppGalNAcT-14 or ppGalNAcT-3 in PSN-1 or DLD-1 cells after 48 hours siRNA knockdown by Taqman assay. (B) GalNAcT-14 (GalNAcT-14 si (1) containing an empty plasmid, wild type GalNAcT-14 (GalNAcT-14), or siGalNAcT-14 (1) mediated knockdown of ppGalNAcT-14 followed by siRNA silent mutations Mut) transfection reconstructs GalNAcT-14 expression in PSN-1 cells. (C) Downregulation of ppGalNAcT-3 or Fut-6 by interfering RNA inhibits Apo2L / TRAIL induced cell death in C170 (colorectal cancer) cells. Experimental procedure as in 11C. (Table 1) A) Summary table of siRNA knockdown phenotypes. Cell lines where downregulation of GalNAcT-14 or ppGalNAcT-3 and Fut-6 resulted in protection from Apo2L / TRAIL protected less than 50% (+) or more (++) with one or more tested siRNA oligonucleotides Is displayed while pointing to. (0) indicates the absence of protection against Apo2L / TRAIL. Following (48) knockdown to (D), (E) indicated siRNAs, cells were treated with increasing doses of etoposide or staurosporin (STS) for 24 hours and subjected to cell viability assays. (F) Retroviral ppGalNAcT-14 overexpressing PA-TU-8902 and PL-45 cell line pools after Apo2L / TRAIL treatment were subjected to cell viability assay. Western blot analysis using anti-FLAG antibodies indicated retroviral expression ppGalNAcT-14 in these cells.

Figure 15 (A) Western blot analysis of Apo2L / TRAIL-induced caspase activation cascade in Apo2L / TRAIL sensitive Colo205 and resistant colorectal cancer cell lines RKO and SW1417. Cells were treated with 1000 ng / ml Apo2L / TRAIL for 8 and 24 hours, and total cell lysates were treated with caspase-8, Bid, caspase-9, caspase-3 and actin specific antibodies as loading controls. It was applied to the western blot analysis used. (B) Knockdown of Fut-6 reduced the recruitment and activation of caspase-8 in Apo2L / TRAIL DISC in DLD-1 cells. Experimental procedure according to 12D. (C) Cell surface expression of DR4 and DR5 was measured by FACS analysis in cells subjected to siRNA knockdown to the indicated genes.

Techniques and procedures described or referenced herein are generally well understood by those of ordinary skill in the art, and are routine methodologies, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.], and is commonly used using the widely used molecular cloning methodology. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to protocols and / or parameters as specified by the manufacturer unless otherwise noted.

Before the methods and assays of the present invention are described, it should be understood that the present invention is not limited to particular methodologies, protocols, cell lines, animal species or genus, constructs, and reagents, as these may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

It should be noted that the indefinite articles and definite articles used in this specification and the appended claims include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “gene modifications” includes many such modifications, and reference to “probes” refers to one or more probes and their equivalents known to those skilled in the art.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in which the publications are cited. Publications cited herein are cited for the disclosure of the publication prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors do not have the right to prioritize the publication by date of priority or priority of the invention. In addition, the actual publication date may be different from that presented and may require separate confirmation.

I. Definition

The terms "Apo2L / TRAIL", "Apo-2L" and "TRAIL" refer to amino acid residues 114-281 (including boundaries), 95-281 (including boundaries), residues 92-281 (including boundaries) of the amino acid sequence shown in FIG. , A polypeptide sequence comprising residues 91-281 (including boundaries), residues 41-281 (including boundaries), residues 15-281 (including boundaries), or residues 1-281 (including boundaries), as well as biologically It is used herein to refer to active fragments, deletions, insertions or substitution variants. In one embodiment, the polypeptide sequence comprises residues 114-281 of FIG. 1 and optionally consists of residues 114-281 of FIG. 1. Optionally, the polypeptide sequence comprises residues 92-281 or residues 91-281 of FIG. 1. Apo-2L polypeptide may be encoded by the native nucleotide sequence shown in FIG. 1. Optionally, the codon encoding residue Pro119 (FIG. 1) can be “CCT” or “CCG”. In another embodiment, the fragment or variant is biologically active and has at least about 80%, more preferably at least about 90%, even more preferably amino acid sequence identity with any one of the Apo2L / TRAIL sequences listed above Is at least 95%, 96%, 97%, 98%, or 99%. Optionally, Apo2L / TRAIL polypeptides are encoded by nucleotide sequences that hybridize with the coding polynucleotide sequences provided in FIG. 1 under stringent conditions. The definition includes substitutional variants of Apo2L / TRAIL in which at least one of the natural amino acids of Apo2L / TRAIL is substituted with an alanine residue. Certain substitution variants of Apo2L / TRAIL include those in which one or more amino acids are substituted by an alanine residue. Such substitutional variants include, for example, those identified as "D203A", "D218A" and "D269A". This nomenclature is used to identify Apo2L / TRAIL variants in which aspartic acid residues at positions 203, 218 and / or 269 (using the numbering set forth in FIG. 1) are substituted with alanine residues. Optionally, the Apo2L variant may comprise one or more of the alanine substitutions listed in Table I of published PCT application WO 01/00832. Substitution variants include one or more of the residue substitutions identified in Table I of WO 01/00832 (published Jan. 4, 2001). The definition also includes the native sequence Apo2L / TRAIL isolated from an Apo2L / TRAIL source or prepared by recombinant or synthetic methods. Apo2L / TRAIL of the present invention includes polypeptides referred to as Apo2L / TRAIL or TRAIL as disclosed in PCT Publications WO 97/01633 and WO 97/25428. The term “Apo2L / TRAIL” or “Apo2L” is used to generally refer to a form of Apo2L / TRAIL, including monomeric, dimeric or trimeric forms of the polypeptide. All numbering of amino acid residues referred to in the Apo2L sequence uses numbering according to FIG. 1, unless explicitly stated otherwise. For example, "D203" or "Asp203" refers to the aspartic acid residue at position 203 in the sequence provided in FIG.

The term "Apo2L / TRAIL extracellular domain" or "Apo2L / TRAIL ECD" refers to a form of Apo2L / TRAIL that is essentially free of transmembrane and cytoplasmic domains. Typically, the ECD will have less than 1% such transmembrane domains and cytoplasmic domains, preferably less than 0.5% such domains. It will be understood that all transmembrane domain (s) identified for the polypeptides of the invention are identified according to the criteria routinely used in the art to identify hydrophobic domains of this type. The exact boundaries of the transmembrane domain may change, but will probably change by up to about 5 amino acids at either end of the initially identified domain. In a preferred embodiment, the ECD will consist of the soluble extracellular domain sequence of the polypeptide without the transmembrane domain and the cytoplasmic or intracellular domain (and not bound to the membrane). Specific extracellular domain sequences of Apo-2L / TRAIL are described in PCT Publications WO 97/01633 and WO 97/25428.

The term "Apo2L / TRAIL monomer" or "Apo2L monomer" refers to the covalent chain of the extracellular domain sequence of Apo2L.

The term “Apo2L / TRAIL dimer” or “Apo2L dimer” refers to two Apo-2L monomers covalently linked via disulfide bonds. As used herein, the term includes free Apo2L dimers and Apo2L dimers (ie associated with another third Apo2L monomer) present in the trimeric form of Apo2L.

The term "Apo2L / TRAIL trimer" or "Apo2L trimer" refers to three Apo2L monomers associated in a non-covalent association.

The term “Apo2L / TRAIL aggregate” is used to refer to a self-associated higher oligomeric form of Apo2L / TRAIL, such as Apo2L / TRAIL trimer, which forms, for example, the hexameric and tetrameric forms of Apo2L / TRAIL. . Determination of the presence and amount of Apo2L / TRAIL monomers, dimers, or trimers (or other aggregates) using methods and assays known in the art, such as natural size exclusion HPLC ("SEC"), sodium dodecyl sulfate Denaturation size exclusion (“SDS-SEC”), reverse phase HPLC and capillary electrophoresis (and using commercially available materials).

“Apo-2 ligand receptor” includes receptors referred to in the art as “DR4” and “DR5”, the polynucleotide and polypeptide sequences thereof are shown in FIGS. 2 and 3, respectively. Pan et al., Science, 276: 111-113 (1997) describe TNF receptor family members referred to as “DR4” (also WO 98/32856 (published 30 July 1998); WO 99 / 37684 (published 29 July 1999); WO 00/73349 (published 7 December 2000); US 6,433,147 (August 13, 2002); US 6,461,823 (October 8, 2002) and US 6,342,383 (January 29, 2002). Another receptor for Apo2L / TRAIL is described in Sheridan et al., Science, 277: 818-821 (1997) and Pan et al., Science, 277: 815-818 (1997). WO 98/51793 published November 19, 1998; WO 98/41629 published September 24, 1998). This receptor is referred to as DR5 (this receptor is alternatively also referred to as Apo-2; TRAIL-R, TR6, Tango-63, hAPO8, TRICK2 or KILLER; Screaton et al., Curr. Biol., 7 : 693-696 (1997); Walczak et al., EMBO J., 16: 5386-5387 (1997); Wu et al., Nature Genetics, 17: 141-143 (1997); WO 98 / 35986 (published August 20, 1998); EP 870,827 (published October 14, 1998); WO 98/46643 (published October 22, 1998); WO 99/02653 (published January 21, 1999) WO 99/09165 published 25 February 1999, WO 99/11791 published March 11, 1999; US 2002/0072091 published 13 August 2002; US 2002/0098550 (2001) Published 7 December); US 6,313,269 issued December 6, 2001; US 2001/0010924 published August 2, 2001; US 2003/01255540 published 3 July 2003; US 2002 / 0160446 published 31 October 2002, US 2002/0048785 published 25 April 2002, US 6,569,642 issued May 27, 2003 US 6,072,047 issued June 6, 2000, US 6,642,358 issued November 4, 2003). As described above, other receptors for Apo-2L include DcR1, DcR2 and OPG (Sheridan et al., Supra), Marsters et al., Supra and Simonet et al., Supra Literature]. The term "Apo-2L receptor" as used herein includes native sequence receptors and receptor variants. This term includes Apo-2L receptors expressed in various mammals, including humans. Apo-2L receptors can be expressed endogenously as occurs naturally in a variety of human tissue lineages, or can be expressed by recombinant or synthetic methods. "Native sequence Apo-2L receptor" includes polypeptides having the same amino acid sequence as the naturally occurring Apo-2L receptor. Thus, the native sequence Apo-2L receptor can have the amino acid sequence of naturally occurring Apo-2L receptor from any mammal. Such native sequence Apo-2L receptors can be isolated from nature or can be produced by recombinant or synthetic means. The term “natural sequence Apo-2L receptor” refers to a naturally-occurring truncated or secreted form of a receptor (eg, a soluble form that contains, eg, an extracellular domain sequence), a naturally-occurring variant form (eg, alternating) Spliced forms) and naturally-occurring allelic variants in particular. Receptor variants may include fragments or deletion mutants of the native sequence Apo-2L receptor. 3A shows the sequence of 411 amino acids of human DR5 as disclosed in WO 98/51793 (November 19, 1998 published). Transcription splice variants of human DR5 are known in the art. This DR5 splice variant encodes the sequence of 440 amino acids of human DR5 as shown in FIGS. 3B and 3C as published in WO 98/35986 (August 20, 1998 publication).

“Death receptor antibody” is used herein to generally refer to an antibody or antibodies that are directed against a receptor of the tumor necrosis factor receptor superfamily and that include a death domain capable of delivering signals of apoptosis, such antibodies are DR5 antibodies and DR4. Antibody.

A "DR5 receptor antibody", "DR5 antibody" or "anti-DR5 antibody" is one or more forms of DR5 receptor, such as the 1-411 sequence shown in Figure 3A or the 1-440 sequence shown in Figures 3B-3C, or a cell thereof. It is used in a broad sense to refer to an antibody that binds to a foreign domain. Optionally, the DR5 antibody is fused or linked to a heterologous sequence or molecule. Preferably, the heterologous sequence allows or assists the antibody to form higher dimensional or oligomeric complexes. Optionally, the DR5 antibody binds to the DR5 receptor but does not bind or cross react with any additional Apo-2L receptor (eg, DR4, DcR1 or DcR2). Optionally, the antibody is an agonist of DR5 signaling activity.

Optionally, the DR5 antibodies of the invention bind to the DR5 receptor at a concentration of about 0.1 nM to about 20 mM as measured by the BIAcore binding assay. Optionally, the DR5 antibodies of the invention exhibit an IC50 value of about 0.6 nM to about 18 mM as measured by the BIAcore binding assay.

"DR4 receptor antibody", "DR4 antibody" or "anti-DR4 antibody" is used in a broad sense to refer to an antibody that binds one or more forms of the DR4 receptor or its extracellular domain. Optionally, the DR4 antibody is fused or linked to a heterologous sequence or molecule. Preferably, the heterologous sequence allows or assists the antibody to form higher dimensional or oligomeric complexes. Optionally, the DR4 antibody binds to the DR4 receptor but does not bind or cross react with any additional Apo-2L receptor (eg, DR5, DcR1 or DcR2). Optionally, the antibody is an agonist of DR4 signaling activity.

Optionally, the DR4 antibodies of the invention bind to the DR4 receptor at a concentration of about 0.1 nM to about 20 mM as measured by the BIAcore binding assay. Optionally, the DR4 antibodies of the invention exhibit an IC50 value of about 0.6 nM to about 18 mM as measured by the BIAcore binding assay.

The term “agonist” is used in its broadest sense and partially or wholly enhances, stimulates or enhances one or more biological activities of Apo2L / TRAIL, DR4 or DR5 in vitro, in situ or in vivo. It includes any molecule that activates. Examples of such biological activities are the binding of Apo2L / TRAIL to DR4 or DR5, including those reported in the literature as well as apoptosis. The agonist may function in a direct or indirect manner. For example, an agonist can partially or wholly complete one or more biological activities of DR4 or DR5 in vitro, in situ or in vivo as a result of direct binding to DR4 or DR5 resulting in receptor activation or signal transduction. Function to augment, stimulate or activate. An agonist may also be indirectly in vitro, in situ or in vivo, for example, as a result of stimulating another effector molecule, which in turn results in DR4 or DR5 activation or signal transduction. May function to partially, fully enhance, stimulate, or activate one or more biological activities of DR4 or DR5. It is contemplated that an agonist may act as an enhancer molecule that functions to indirectly enhance or increase DR4 or DR5 activation or activity. For example, agonists can enhance the activity of endogenous Apo-2L in mammals. This can be achieved, for example, by pre-complexing DR4 or DR5 or by stabilizing the complex of the DR4 or DR5 receptor with each ligand (eg by stabilizing the native complex formed between Apo-2L and DR4 or DR5). have.

As used herein, the term “biomarker” can detect expression in or on mammalian tissue or cells by standard methods (or methods disclosed herein), and such detection is directed to Apo2L / TRAIL or killed receptor antibodies. A molecule generally comprising a gene, protein, carbohydrate structure, or glycolipid, which predicts the sensitivity of a mammalian cell or tissue. Such biomarkers embodied by the present invention include, but are not limited to, molecules belonging to proteins of the GalNac-T family. Members of genes and proteins of the human N-acetylgalactosaminiltransferase ("GalNac-T") family have been described (see, eg, Hang et al., "The chemistry and biology of mucin-type O- linked glycosylation initiated by the polypetide N-acetyl- -galactosaminyltransferases ", Bioorganic & Medicinal Chemistry (available at www.sciencedirect.com in May 2005) and references cited therein; Wang et al., BBRC, 300: 738-744 (2003) and references cited therein), which are believed to function in determining the number and location of O-linked sugar chains in a protein. Optionally, the expression of such biomarkers is determined to be higher than that observed for control tissue or cell samples. Optionally, for example, expression of such biomarkers will be determined using gene expression microarray, quantitative PCR or immunohistochemistry (IHC) assay. Optionally, the expression of the GalNac-T biomarker, such as GalNac-T14 or GalNac-T3, is greater in the test tissue or cell sample than was observed for the control tissue or cell sample when detecting expression of the biomarker using quantitative PCR, When measured by Affymetrix U133P microarray analysis, it will be detected at a level of at least 750 or 500 times, preferably at least 1000 times higher.

"UDP-N-acetyl-D-galactosamine polypeptide N-acetylgalactosamineyltransferase-T14", "pp-GalNac-T14", "GalNac-T14", "GALNT14" are N-terminal cytoplasmic domain, membrane It is used herein to refer to a type II membrane protein having the characteristic features of a GalNac-T group molecule comprising a transverse domain, a stem region and a catalytic domain. In certain embodiments, the human GalNac-T14 molecule contains 1659 base pairs that encode a protein of 552 amino acids, as shown in FIGS. 4A1-4A4. Full length human cDNA was deposited with GenBank under accession number AB078144. As described in Wang et al., BBRC, 300: 738-744 (2003), a subset of GalNac-T14 containing (or not including) certain exons, such as exons 2, 3, and / or 4 Flying isoforms have been identified. The present invention embodies an investigation of the expression of any such various isotypes of GalNac-T14, and the expression of any one such isotype predicts the sensitivity of a mammalian or cell sample to Apo2L / TRAIL or a death receptor antibody. .

"UDP-N-acetyl-D-galactosamine polypeptide N-acetylgalactosaminiltransferase-T3", "pp-GalNac-T3", "GalNac-T3", "GALNT3" are N-terminal cytoplasmic domain, membrane It is used herein to refer to a type II membrane protein having the characteristic features of a GalNac-T group molecule comprising a transverse domain, a stem region and a catalytic domain. In certain embodiments, human GalNac-T3 polypeptide comprises the amino acid sequence shown in FIGS. 4B1-4B4. GalNac-T3 is described by Bennett et al., J. Biol. Chemistry, 271: 17006-17012 (1996).

"Subject" or "patient" refers to any single subject for which treatment is desired, including humans. Any subject included in clinical research trials that do not exhibit any clinical signs of disease, or subjects included in epidemiological studies, or subjects used as controls, are also intended to be included.

As used herein, the term “mammal” refers to any mammal classified as a mammal, including humans, cattle, horses, dogs, and cats. In a preferred embodiment of the invention, the mammal is a human.

"Tissue or cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. The source of tissue or cell sample may be solid tissue from a fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood component; Body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; Cells from any time during pregnancy or development of the subject. The tissue sample can also be primary or cultured cells or cell lines. Optionally, tissue or cell samples are obtained from primary or metastatic tumors. Tissue samples may contain compounds that are not inherently mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

For the purposes herein, “fragment” of a tissue sample means a single portion of a tissue sample or a slice of tissue, eg, tissue or cells cut from a tissue sample. As long as the present invention is understood to include a method in which the same segment of a tissue sample is analyzed at both morphological and molecular levels or for both protein and nucleic acid, multiple sections of tissue samples are taken and applied to the assay according to the invention. It is understood that it can be done.

By “correlating” or “correlating” is meant to compare the performance and / or results of the first analysis or protocol in any way with the performance and / or results of the second analysis or protocol. For example, the results of the first analysis or protocol can be used to perform the second protocol and / or the results of the first analysis or protocol can be used to determine whether the second analysis or protocol should be performed. In connection with various embodiments herein, analytical assays, such as the results of mRNA expression or IHC, can be used to determine whether a particular therapy using Apo2L / TRAIL or a death receptor antibody should be performed.

"Nucleic acid" is intended to include any DNA or RNA. For example, chromosomes, mitochondria, viral and / or bacterial nucleic acids are present in tissue samples. The term “nucleic acid” includes one or both strands of a double stranded nucleic acid molecule and includes any fragment or portion of an intact nucleic acid molecule.

"Gene" refers to any nucleic acid sequence or portion thereof that has a functional role in the coding or transcription of proteins or the regulation of other gene expression. The gene may consist of all nucleic acids responsible for coding the functional protein, or may consist only of a portion of the nucleic acid responsible for protein coding or expression. Nucleic acid sequences may contain gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique regions adjacent to genes.

As used herein, the word “label” refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent, such as a nucleic acid probe or antibody, and that facilitates detection of a reagent to which a label is conjugated or fused. The label may itself be detectable (eg a radioisotope label or fluorescent label), or in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.

As used herein, the term "antibody" is used in its broadest sense and is specifically an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from two or more intact antibodies (eg, Bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity.

An "antibody fragment" includes a portion of an intact antibody, and preferably includes an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments; Diabody; Linear antibodies; Single chain antibody molecules; And multispecific antibodies formed from antibody fragments.

Generally “natural antibodies” are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide linkages differs between the heavy chains of several immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end; The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

The term “variable” refers to the fact that certain portions of the variable domains differ greatly in sequence between antibodies and are used in the binding and specificity of each particular antibody to its specific antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called hypervariable regions or complementarity determining regions in both the light and heavy chain variable domains. The more highly conserved portions in the variable domains are called the framework regions (FRs). The variable domains of each of the natural heavy and light chains are mainly adopting a β-sheet shape, linked by three hypervariable regions that form a loop that forms a part of the β-sheet structure, in some cases 4. Two FRs. The hypervariable regions in each chain are held together very closely by the FRs and contribute to forming the antigen-binding site of the antibody with the hypervariable regions from the other chain (Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD (1991). The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions such as the involvement of the antibody in antibody dependent cell type cytotoxicity (ADCC).

Digestion of the antibody with papain results in two identical antigen-binding fragments, each with a single antigen-binding site, referred to as a "Fab" fragment, and the remaining "Fc" fragment, which is easily crystallized. It is a reflection of ability. Treatment with pepsin yields an F (ab ') ₂ fragment that has two antigen binding sites and is still capable of crosslinking with the antigen.

"Fv" is the minimum antibody fragment which contains a complete antigen-recognition site and an antigen-binding site. This region consists of a dimer where one heavy chain variable domain and one light chain variable domain are associated in tight non-covalent association. It is within this shape that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V _H -V _L dimer. Collectively, six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv including only three hypervariable regions specific for the antigen), although having a lower affinity than the entire binding site, has the ability to recognize and bind antigen.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain comprising one or more cysteines from the antibody hinge region. Fab'-SH is the name herein for Fab 'in which the cysteine residue (s) of the constant domains have one or more free thiol groups. F (ab ') ₂ antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

The “light chain” of an antibody (immunoglobulin) from any vertebrate species can be assigned to one of two distinctly different types, called kappa (κ) and lambda (λ), based on the amino acid sequences of the constant domains. .

Depending on the amino acid sequence of the constant domain of the "heavy chain", antibodies can be assigned to several classes. Intact antibodies have five major classes of IgA, IgD, IgE, IgG and IgM, some of which are further classified as subclasses (isotypes), for example IgG1, IgG2, IgG3, IgG4, IgA and IgA2. Can be. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

"Single-chain Fv" or "scFv" antibody fragments comprise the V _H and V _L antibody domains of an antibody present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V _H and V _L domains which enables the scFv to form the desired structure for antigen binding. For an overview of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "dia body" in the same polypeptide chain, the light chain variable domain (V _L) chain variable domain (V _H), 2 different antigens, including linked to in (V _H -V _L) - refers to a small antibody fragments with a binding site do. By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with the complementary domain of another chain, resulting in two antigen-binding sites. Diabodies are described, for example, in EP 404,097; WO 93/11161; And Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are directed against a single antigenic site and are highly specific. In addition, unlike conventional (polyclonal) antibody preparations that typically include several antibodies directed against several determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture that is not contaminated with other immunoglobulins. The modifier “monoclonal” refers to the characteristics of an antibody that is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring antibody production by any particular method. For example, the monoclonal antibodies used according to the present invention can be prepared by the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods ( For example, US Pat. No. 4,816,567). Also "monoclonal antibodies" are described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and in Marks et al., J. Mol. Biol., 222: 581-597 (1991), can be isolated from phage antibody libraries.

Monoclonal antibodies herein include those in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain (s) Particularly included are “chimeric” antibodies (immunoglobulins) identical or homologous to the corresponding sequences of antibodies from other species or belonging to another antibody class or subclass, as well as fragments of such antibodies as long as they exhibit the desired biological activity ( U.S. Patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). Such chimeric antibodies herein include a variable domain antigen-binding sequence and a human constant region sequence derived from a non-human primate (eg, a continental monkey such as baboon, rhesus monkey or cynomolgus monkey). Primateized "antibodies are included (US Pat. No. 5,693,780).

A “humanized” form of a non-human (eg murine) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. For most parts, the humanized antibody may be a hypervariable region (donor antibody) of a non-human species such as a mouse, rat, rabbit or non-human primate whose residues from the hypervariable region of the recipient have the desired specificity, affinity and ability. Human immunoglobulin (receptor antibody) replaced with a residue from In some cases, framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Humanized antibodies may also include residues that are not found in the recipient antibody or in the donor antibody. Such modifications are made to further refine the performance of the antibody. In general, humanized antibodies comprise substantially all of one or more, typically two, variable domains, where all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all or substantially As such all FRs are of human immunoglobulin sequence. Humanized antibodies will also optionally optionally include at least a portion of an immunoglobulin constant region (Fc), typically of human immunoglobulins. Further details are given by Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); And Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

As used herein, the term “hypervariable region” refers to an amino acid residue of an antibody that is responsible for antigen binding. Hypervariable regions include amino acid residues from “complementarity determining regions” or “CDRs” (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3), and heavy chains in the light chain variable domain) 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda (MD. (1991))) and / or residues from “hypervariable loops” (eg, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain) and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

Antibodies that “bind” to an antigen are those capable of binding such antigen with sufficient affinity and / or binding force such that the antibody is useful as a therapeutic or diagnostic agent for targeting cells expressing the antigen.

For the purposes of the present invention, "immunotherapy" will refer to a method of treating a mammal (preferably a human patient) with an antibody, wherein the antibody can be an unconjugated or "naked" antibody. Or an antibody may be conjugated or fused with a heterologous molecule (s) or agent (s), eg, one or more cytotoxic agent (s), to produce an “immunoconjugate”.

An "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are substances that prevent antibodies from being used for diagnosis or treatment and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is (1) in excess of 95% by weight antibody, most preferably in excess of 99% by weight, as measured by the Lowry method, by (2) using a spinning cup sequencer. Sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) until homogeneous by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Will be. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by one or more purification steps.

The expression “effective amount” refers to the amount of an agent (eg, Apo2L / TRAIL, anti-DR4 or DR5 antibody, etc.) effective for preventing, alleviating or treating a disease or condition in question.

As used herein, the terms “treating”, “treatment” or “therapy” refer to healing, prophylactic and prophylactic therapies. Continuous treatment or administration refers to treatment based on at least daily, without one or more treatment interruptions. Intermittent treatment or administration, or intermittent treatment or administration, is not continuous, but refers essentially to periodic treatment.

The term “cytokine” is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokine, monocaine and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; Parathyroid hormone; Thyroxine; insulin; Proinsulin; Relaxin; Prolylacin; Glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); Liver growth factor; Fibroblast growth factor; Prolactin; Placental lactogen; Tumor necrosis factor-α and tumor necrosis factor-β; Muller-inhibiting substance; Mouse gonadotropin-associated peptide; Inhivin; Actibin; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factor; Platelet growth factor; Transforming growth factors (TGF) such as TGF-α and TGF-β; Insulin-like growth factor-I and insulin-like growth factor-II; Erythropoietin (EPO); Osteoinduction factors; Interferons such as interferon-α, interferon-β and interferon-gamma; Colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); Granulocyte-CSF (G-CSF); Interleukins (IL) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; And other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or proteins from recombinant cell culture, and biologically active equivalents of native sequence cytokines.

As used herein, the term “cytotoxic agent” refers to a substance that inhibits or prevents the function of a cell and / or causes cell destruction. The term refers to radioactive isotopes (eg, I ¹³¹ , I ¹²⁵ , Y ⁹⁰ and Re ¹⁸⁶ ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. It is intended to include.

“Chemotherapeutic agents” are chemical compounds useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN ™); Alkyl sulfonates such as busulfan, impprosulfan and pifosulfan; Aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; Ethyleneimine and methyleneimine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylololomeramine; Acetogenin (particularly bulatacin and bulatacinone); Camptothecins (including the synthetic analog topotecan); Bryostatin; Calistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); Cryptopycin (particularly cryptotopin 1 and cryptotopin 8); Dolastatin; Duocarmycin (including the synthetic analogues KW-2189 and CBI-TMI); Eleuterobins; Pankratisstatin; Sarcodictin; Spongestatin; Nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, esturamustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, normovicin, fensterrin, fred Nimustine, trophosphamide, uracil mustard; Nitrosureas such as carmustine, chlorozotocin, potemustine, lomustine, nimustine, rannimustine; Antibiotics such as endynein antibiotics (e.g., calicheamicins, especially calicheamicin gamma1I and calicheamycin piI1 (see, eg, Agnew, Chem Intl. Ed. Engl., 33: 183-186) 1994)]; dynemycin, including dynemycin A; bisphosphonates, such as clodronate; esperamycin; as well as neocarcinonostatin chromophores and related chromoprotein endyne antibiotic chromophores), aclacinomycin , Actinomycin, Otramycin, Azaserine, Bleomycin, Cocktinomycin, Carabicin, Carminomycin, Carcinophylline, Chromomycinis, Dactinomycin, Daunorubicin, Detorrubicin, 6- Diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN ™) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, Esorubicin, this Darvisin, marcelomycin, mitomycin, such as mitomycin C, mycophenolic acid, nogalamycin, olibomycin, peplomycin, potyrromycin, puromycin, kelamycin, rhorubicin, streptonicin, streptozocin , Tubercidine, ubenimex, ginostatin, zorubicin; Anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs such as denophtherine, methotrexate, putrophtherin, trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluidine, enositabine, phloxuridine; Androgens such as calusosterone, dromostanolone propionate, epithiostanol, mepitiostane, or testosterone; Anti-adrenal agents such as aminoglutetimide, mitotan, trilostane; Folic acid supplements such as proline acid; Aceglaton; Aldophosphamide glycosides; Aminolevulinic acid; Eniluracil; Amsacrine; Vestravusyl; Bisantrene; Etrexate; Depopamine; Demecolsin; Diaziquiones; Elponnitine; Elftinium acetate; Epothilones; Etogluside; Gallium nitrate; Hydroxyurea; Lentinane; Ronidinin; Maytansinoids such as maytansine and ansamitosine; Mitoguazone; Mitoxantrone; Fur mall; Nitracrine; Pentostatin; Penammet; Pyrarubicin; Rosantanone; Grape filinic acid; 2-ethylhydrazide; Procarbazine; PSK®; Lakamic acid; Liqin; Sizopyran; Spirogermanium; Tenuazone acid; Triaziquion; 2,2 ', 2 "-trichlorotriethylamine; tricothecene (especially T-2 toxin, veracrine A, loridine A and anguidine); urethane; bindecine; dacarbazine; mannosemusine; mitobronitol; Mitollactol; fifobroman; azaytocin; arabinoxide (“Ara-C”); cyclophosphamide; thiotepa; taxoids such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology (Princeton, NJ)) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer (Antony, France)); chlorambucil; gemcitabine (Gemzar ™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and Carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Iphosphamide; Mitoxantrone; Vincristine; Vinorelbin (Navelbine ™); Novantrone; Teniposide; Edatrexate; Daunooma Lysine; aminopterin; geloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylor Tin (DMFO), retinoids such as retinoic acid, capecitabine, and pharmaceutically acceptable salts, acids or derivatives of any of the above substances, the definitions of which act to modulate or inhibit hormonal action on the tumor. Anti-hormonal agents such as tamoxifen (including Nolvadex ™), raloxifene, droroxifene, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onafristone and toremiphene (Fareston ™) are for example Included anti-estrogen agents and selective estrogen receptor modulators (SERM); aromatase inhibitors that inhibit aromatase, an enzyme that modulates estrogen production in the adrenal glands, such as 4 (5) -imidazole, aminoglu Tetimide, megestrol acetate (Megace ™), exemestane, formestanier, padrosol, borazole (Rivisor ™), letrozole (Femara ™), and anastrozole (Arimidex ™); and anti -And Genje, for example, flutamide, imide carbonyl rutile, rutile Baikal imide, leuprolide, and goserelin; And pharmaceutically acceptable salts, acids or derivatives of any of the above materials.

As used herein, “growth inhibitor” refers to a compound or composition that inhibits the growth of cells in vitro or in vivo, particularly cancer cells that overexpress any of the genes identified herein. Thus, growth inhibitory agents significantly reduce the percentage of cells that overexpress such genes in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (in steps other than S phase), such as agents that induce G1 arrest and M-phase arrest. Traditional M-stage blockers include vinca (vincristine and vinblastine), taxol and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. Agents that stop G1, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also overflow with S-phase stops. For further information, see The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, "Cell cycle regulation, oncogenes, and antineoplastic drugs", Murakami et al. (WB Saunders: Philadelphia, 1995), especially on page 13.

The terms "apoptotic" and "apoptotic activity" are used in a broad sense and include one or more characteristic cellular changes, including cytoplasmic condensation, loss of plasma microvilli, nuclear fission, chromosomal DNA degradation, or loss of mitochondrial function. Typically accompanied by a regular or controlled form of cell death in a mammal. Such activity is described, for example, in cell viability assays (such as Alamar blue assays or MTT assays), FACS assays, caspase activation, DNA fragmentation (eg, as described in Nicoletti et al. , J. Immunol.Methods, 139: 271-279 (1991)), and poly-ADP ribose polymerase ("PARP") cleavage assays.

As used herein, the term “disorder” refers to from treatment with a composition described herein, including any disease or disorder that may be treated with an effective amount of Apo2L / TRAIL, anti-DR4 antibody, and / or anti-DR5 antibody. It generally refers to any condition that would benefit. This includes chronic and acute disorders, as well as pathological conditions that predispose mammals to the disorder in question. Non-limiting examples of disorders to be treated herein include benign and malignant cancers; Inflammatory, angiogenic and immunological disorders, autoimmune disorders, arthritis (including rheumatoid arthritis), multiple sclerosis, and HIV / AIDS.

The term "cancer", "cancerous" or "malignant" refers to or describes a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal cancer (gastrointestinal cancer), kidney cancer, ovarian cancer, Liver cancer, lymphocytic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, bladder cancer, Liver cancer, breast cancer, colon carcinoma, and head and neck cancer.

The term "immune related disease" means a disease in which a component of the mammal's immune system causes, mediates or otherwise contributes to the disease of the mammal. Also included are diseases in which stimulation or intervention of the immune response has an improving effect on disease progression. Such terms include autoimmune diseases, immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, and immunodeficiency diseases. Examples of immune related and inflammatory diseases (some of which are immune or T cell mediated) that can be treated according to the invention include systemic lupus erythematosus, rheumatoid arthritis, childhood chronic arthritis, spondyloarthropathy, systemic sclerosis (skin sclerosis), Idiopathic inflammatory myopathy (skin myositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune thrombocytopenia, paroxysmal nocturnal thrombocytopenia), autoimmune thrombocytopenia, immuno-mediated thrombocytopenia Hypothyroidism), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated kidney disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous system, such as Multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory Myeloid polyneuropathy, hepatobiliary diseases such as infectious hepatitis (A, B, C, D, E and other non-hepatophilic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and Sclerotic cholangitis, inflammatory and fibrotic lung diseases such as inflammatory bowel disease (ulcerative colitis: Crohn's disease); Gluten-sensitive enteropathy, and Whipple disease, autoimmune or immune-mediated skin diseases (including bullous skin disease, polymorphic erythema and contact dermatitis), psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic Dermatitis, food intolerance and urticaria, pulmonary immunological diseases such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and irritable pneumonia, transplant related diseases (graft rejection and graft-versus-host disease). Infectious diseases include AIDS (HIV infection), hepatitis A, B, C, D and E, bacterial infections, fungal infections, protozoal infections and parasitic infections.

"Autoimmune disease" is used herein in a broad, general sense to refer to a disorder or condition in a mammal in which destruction of normal or healthy tissue occurs from a humoral or cellular immune response to an individual mammal's tissue components thereof. do. Examples include but are not limited to lupus erythematosus, thyroiditis, rheumatoid arthritis, psoriasis, multiple sclerosis, autoimmune diabetes, and inflammatory bowel disease (IBD).

As used herein, the term “tagged” refers to a chimeric molecule comprising an antibody or polypeptide fused to a “tag polypeptide”. Tag polypeptides have enough residues to provide epitopes from which antibodies can be made to epitopes, or to provide some other function, such as the ability to oligomerize (eg, occurring in peptides with leucine zipper domains). And is short enough to generally not interfere with the activity of the antibody or polypeptide. Also preferably the tag polypeptide is quite unique so that the tag-specific antibody does not substantially cross react with other epitopes. Suitable tag polypeptides generally have six or more amino acid residues, usually between about 8 and about 50 amino acid residues (preferably between about 10 and about 20 residues).

The term "divalent metal ion" refers to a metal ion having two positive charges. Examples of divalent metal ions include, but are not limited to, zinc, cobalt, nickel, cadmium, magnesium and manganese. Specific forms of such metals that may be used include salt forms (eg, pharmaceutically acceptable salt forms) such as the chloride, acetate, carbonate, citrate and sulfate forms of the divalent metal ions mentioned above. . Optionally, the divalent metal ion for use in the present invention is zinc, preferably zinc sulfate or zinc chloride in salt form.

"Isolated", when used to describe the various proteins disclosed herein, means proteins identified and separated and / or recovered from components of their natural environment. Contaminant components of its natural environment are substances that typically prevent the peptide or protein from being used for diagnosis or treatment, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the peptide or protein is sufficient to (1) obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (2) Coomassie blue or preferably silver It will be purified until homogeneous by SDS-PAGE under reducing or non-reducing conditions using staining, or (3) homogeneous by mass spectroscopic or peptide mapping techniques. Isolated material includes peptides or proteins in situ within recombinant cells since at least one component of the natural environment of the peptide or protein will not be present. Ordinarily, however, isolated peptide or protein will be prepared by one or more purification steps.

"Percent amino acid sequence identity (%)" associated with a sequence identified herein refers to a candidate sequence that is identical to the amino acid residue in the reference sequence after aligning the sequences and introducing a gap if necessary to achieve a maximum percentage of sequence identity. It is defined as the percentage of amino acid residues within, wherein no conservative substitutions are considered part of the sequence identity. Alignments aimed at determining the percentage of amino acid sequence identity include a variety of techniques within the art that can determine suitable parameters for measuring alignment, including the assignment algorithm necessary to achieve maximal alignment over the full length of the sequences being compared. It can be achieved in a way. For purposes herein, the amino acid identity percentage value is produced by Genentech, Inc., and the source code is submitted with the user documentation to the United States Copyright Office (Washington, DC, 20559). Can be obtained using the sequence comparison computer program "ALIGN-2" registered under copyright TXU510087. The ALIGN-2 program is available from Genentech, Inc. Publicly available from South San Francisco, CA. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

The "stringency" of the hybridization reaction can be readily determined by one skilled in the art and is generally empirically calculated according to probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of the denatured DNA to anneal again when the complementary strand is in an environment below its melting temperature. The higher the degree of desired identity between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For further details and explanations of the stringency of the hybridization reaction, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"High stringency conditions" as defined herein include (1) using low ionic strength and high temperature for washing; 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C .; (2) using denaturant during hybridization; 50% (v / v) formamide + 0.1% bovine serum albumin / 0.1% picol / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) + 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C .; Or (3) 50% formamide at 42 ° C., 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × denhardt solution, sonicated Salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, washed with 0.2 × SSC (sodium chloride / sodium citrate) at 42 ° C., 50% formamide at 55 ° C., and then 55 This is confirmed by using a high stringency wash consisting of 0.1 x SSC containing EDTA at ° C.

"Moderate stringency conditions" can be identified as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, 20% formamide, 5 x SSC ( In a solution comprising 150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA After incubation at 37 ° C. overnight, the filter is washed at about 37-50 ° C. in 1 × SSC. Those skilled in the art will recognize how to adjust the temperature, ionic strength, etc. as needed to adapt to factors such as probe length and the like.

The term "primer" or "primers" refers to a polynucleotide from a mononucleotide by the action of nucleotidyltransferases, hybridized to complementary RNA or DNA target polynucleotides, such as, for example, in a polymerase chain reaction. Refers to an oligonucleotide sequence that serves as a starting point for the stepwise synthesis of nucleotides.

The term "control sequence" refers to a DNA sequence necessary for the expression of a coding sequence operably linked in a particular host organism. For example, control sequences suitable for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, the DNA for the pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a pre-protein that participates in secretion of the polypeptide; The promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or when the ribosomal binding site is positioned to facilitate translation, it is operably linked to a coding sequence. In general, "operably linked" means that the DNA sequences to which they are linked are contiguous, and in the case of a secretory leader, are contiguous and in reading. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refers to the binding of nonspecific cytotoxic cells (eg, natural killer (NK) cells, neutrophils and macrophages) that express Fc receptors (FcRs) onto target cells. It refers to a cell-mediated response that recognizes the antibody and then causes lysis of the target cell. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is described by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991), is summarized in Table 3 on page 464. To assess ADCC activity of the molecule, in vitro ADCC assays, such as those described in US Pat. No. 5,500,362 or 5,821,337, can be performed. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule can be assessed in vivo, for example in an animal model such as disclosed in Clynes et al., PNAS (USA) 95: 652-656 (1998).

"Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably such cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, with PBMCs and NK cells being preferred.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. Preferred FcRs are naturally-sequence human FcRs. In addition, preferred FcRs are receptors (gamma receptors) that bind IgG antibodies, including receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants of these receptors and alternatively spliced forms. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”), which have similar amino acid sequences that differ mainly in the cytoplasmic domain. Activating receptor FcγRIIA contains a tyrosine-based activation motif (ITAM) in the cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); And de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs that are to be identified later are included in the term "FcR" herein. The term also includes neonatal receptor FcRn, which is responsible for delivering maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24 : 249 (1994)]). FcRs herein include genetic polymorphisms, such as genetic dysplasia in a gene encoding FcγRIIIa, resulting in phenylalanine (F) or valine (V) at amino acid position 158 located in the region of the receptor that binds IgG1. Homozygous valine FcγRIIIa (FcγRIIIa-158V) has a higher affinity for human IgG1 and increased in vitro compared to homozygous phenylalanine FcγRIIIa (FcγRIIIa-158F) or heterozygous (FcγRIIIa-158F / V) receptors. It has been shown to mediate.

"Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to dissolve a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (C1q) of the complement system to a molecule (eg an antibody) complexed with a cognate antigen. To assess complement activation, see, eg, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) can be performed for CDC analysis.

II . Representative Methods and Materials of the Invention

The methods and assays disclosed herein relate to examining expression of one or more biomarkers in a mammalian tissue or cell sample, wherein determination of expression of the one or more such biomarkers is determined by Apo2L / TRAIL and / or the tissue or cell sample. Or predict or be sensitive to agents such as death receptor antibodies, such as anti-DR5 agonist antibodies or anti-DR4 agonist antibodies. Such methods and assays include methods and assays for examining the expression of members of the GalNac-T family of molecules, including GalNac-T14 and GalNac-T3.

As discussed above, there are some populations of disease human cell types that are resistant to the apoptosis inducing effects of Apo2L / TRAIL or death receptor antibodies. Accordingly, it is believed that the disclosed methods and assays may provide a convenient, efficient, and potentially cost-effective means for obtaining data and information useful for evaluating suitable or effective therapies for treating patients. For example, a patient diagnosed with cancer or immune-related conditions may be subjected to a biopsy to obtain a tissue or cell sample, and the sample may be examined by various in vitro assays so that the patient's cells may be Apo2L / TRAIL or death receptors. It may be determined whether it will be sensitive to a therapeutic agent such as an antibody.

The present invention provides a method for predicting the sensitivity of a mammalian tissue or cell sample (such as cancer cells) to Apo2L / TRAIL or death receptor agonist antibodies. Optionally, mammalian tissue or cell samples are obtained and examined for expression of GalNac-T14. This method can be used to detect mRNA expression, assays for detecting protein expression (such as immunohistochemistry) and biochemical assays for detecting enzymatic UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminiltransferase activity. This can be done by a variety of assay formats included. Determination of expression of such GalNac-T14 biomarkers in (or on tissue or cells) the tissues or cells will predict that such tissues or cells will be sensitive to the biological activity of Apo2L / TRAIL and / or death receptor antibodies. Applicants have unexpectedly found that expression of GalNac-T14 correlates with the sensitivity of such tissues or cells to Apo2L / TRAIL and death receptor agonist antibodies.

As discussed below, the expression of various biomarkers, such as GalNac-T14, in a sample can be determined by immunohistochemical and / or Western analysis, blood based quantitative assays (eg serum ELISA) (eg, proteins To investigate expression levels), biochemical enzyme activity assays, in situ hybridization, Northern analysis and / or PCR analysis of mRNA, and genomic Southern analysis (eg, to investigate gene deletion or amplification), as well as genes and And / or can be analyzed by any number of methodologies known in the art and understood by one of ordinary skill in the art, including but not limited to any of a wide variety of assays that can be performed by tissue array analysis. Typical protocols for assessing the status of genes and gene products are described, for example, in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis).

The following protocol regarding the detection of GalNac-T14 in a sample is provided below for illustrative purposes.

Any method of the present invention includes a protocol for examining or testing for the presence of GalNac-T14 in a mammalian tissue or cell sample. Various methods for detecting GalNac-T14 can be used, including, for example, immunohistochemical analysis, immunoprecipitation, Western blot analysis, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting (PACS), and Immunoprecipitation followed by MS, monosaccharide analysis. For example, any method of detecting expression of GalNac-T14 in a tissue or sample includes contacting the sample with an anti-GalNac-T14 antibody and then detecting binding of the antibody to GalNac-T14 in the sample.

In certain embodiments of the invention, expression of GalNac-T14 in the sample is investigated using immunohistochemistry and staining protocols. Immunohistochemical staining of tissue sections has proven to be a reliable method of assessing or detecting the presence of proteins in a sample. Immunohistochemistry (“IHC”) techniques utilize antibodies to probes and generally visualize cellular antigens in situ by color or fluorescent methods.

For sample preparation, tissue or cell samples from mammals (typically human patients) can be used. Examples of samples include, but are not limited to, cancer cells such as colon, breast, prostate, ovary, lung, stomach, pancreas, lymphoma and leukemia cancer cells. Optionally, the sample comprises non-small cell lung cancer cells, pancreatic cancer cells or non-Hodgkin's lymphoma cancer cells. Samples can be obtained by a variety of procedures known in the art, including but not limited to surgical excision, aspiration or biopsy. The tissue may be fresh or frozen. In one embodiment, the sample is fixed and embedded in paraffin or the like.

Tissue samples can be fixed (ie, preserved) by conventional methods (see, eg, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology," 3rd edition (1960) Lee G. Luna, HT ( ASCP) Editor, The Blakston Division McGraw-Hill Book Company, New York ;; The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, DC). Those skilled in the art will appreciate that the fixative is selected for the purpose that the sample is histologically stained or otherwise analyzed. One skilled in the art will also understand that the length of fixation depends on the size of the tissue sample and the fixative used. By way of example, neutral buffered formalin, Bouin's or paraformaldehyde can be used to immobilize the sample.

Generally, the sample is first immobilized and then dehydrated through a series of upward concentrations of alcohol and infiltrated and embedded in paraffin or other fragmentation media to allow for sectioning of the tissue sample. Alternatively, the tissue can be sectioned and the obtained sections can be fixed. By way of example, tissue samples may be embedded in paraffin and processed by conventional methods (see, eg, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", supra). Examples of paraffins that can be used include, but are not limited to, Paraplast, Broloid, and Tissuemay. Once the tissue sample is embedded, the sample can be sectioned by microtome or the like (see, eg, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", supra). As an example of this procedure, the thickness of the sections can range from about 3 microns to about 5 microns. Once sectioned, the sections can be attached to the slides by several standard methods. Examples of slide adhesives include, but are not limited to, silane, gelatin, poly-L-lysine, and the like. By way of example, sections embedded in paraffin may be attached to slides with positive charge and / or slides coated with poly-L-lysine.

When paraffin is used as the embedding material, tissue sections are generally deparaffinized and rehydrated in water. Tissue sections can be deparaffinized by several conventional standard methods. For example, a series of alcohols of xylene and gradual downward concentration can be used (see, eg, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", supra). Alternatively, commercially available deparaffinized non-organic agents such as Hemo-De7 (CMS, Houston, Texas) can be used.

Optionally, following sample preparation, tissue sections can be analyzed using IHC. IHC can be performed in combination with additional techniques such as morphological staining and / or fluorescence in situ hybridization. Two general IHC methods are available; Direct and indirect methods. According to the first assay, the binding of the antibody to the target antigen (eg GalNac-T14) is determined directly. This direct assay uses labeled reagents such as fluorescent tags or enzyme-labeled primary antibodies that can be visualized without further antibody interactions. In a typical indirect assay, the unconjugated primary antibody binds to the antigen and then the labeled secondary antibody binds to the primary antibody. If the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorescent substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody.

Primary and / or secondary antibodies used in immunohistochemistry will typically be labeled with a detectable moiety. Many labels are available and they generally fall into the following categories:

(a) radioisotopes, such as ³⁵ S, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I. See, eg, Current Protocols in Immunology Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, New York, Pubs. (1991) can be used to label antibodies with radioisotopes and the radioactivity can be measured using scintillation counting.

(b) colloidal gold particles.

(c) rare earth chelates (Europium chelates), Texas Red, rhodamine, fluorescein, sweets, lissamine, umbeliferon, phycocritin, phycocyanin, or commercially available fluorophores, Fluorescent labels, including but not limited to SPECTRUM 0RANGE7 and SPECTRUM GREEN7 and / or any one or more derivatives of the foregoing. Fluorescent labels can be conjugated to antibodies using, for example, the techniques disclosed in Current Protocols in Immunology, supra. Fluorescence can be quantified using a fluorometer.

(d) Various enzyme-substrate labels are available and US Pat. No. 4,275,149 provides an overview of some of these. Enzymes generally catalyze the chemical alteration of a chromogenic substrate, which can be measured using a variety of techniques. For example, enzymes can catalyze color changes in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme can alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying the change in fluorescence are described above. The chemiluminescent substrate is electrically excited by a chemical reaction and then emits light that can be measured (eg, using a chemiluminescence analyzer) or energizes the fluorescent receptor. Examples of enzyme labels include luciferase (eg firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidase Such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase) ), Heterocyclic oxidases (eg uricase and xanthine oxidase), lactoperoxidases, microperoxidases and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, Method in Enzym. (ed. J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

Examples of enzyme-substrate combinations include, for example, the following:

(i) horseradish peroxidase (HRPO) and hydrogen peroxidase as a substrate, where hydrogen peroxidase is a dye precursor (e.g. orthophenylene diamine (OPD) or 3,3 ', 5,5'-tetramethyl Oxidize benzidine hydrochloride (TMB));

(ii) alkaline phosphatase (AP) and para-nitrophenyl phosphate as chromogenic substrate; And

(iii) β-D-galactosidase (β-D-Gal) and chromogenic substrates (eg p-nitrophenyl-β-D-galactosidase) or fluorescent substrates (eg 4-methyl Umbeliferyl-β-D-galactosidase).

Many other enzyme-substrate combinations are available to those skilled in the art. For a general overview of these, see US Pat. Nos. 4,275,149 and 4,318,980. Sometimes the label is indirectly conjugated to the antibody. Those skilled in the art will be aware of various techniques for achieving this. For example, the antibody may be conjugated with biotin, and any of the four broad categories of labels mentioned above may be conjugated to avidin, or vice versa. Biotin binds selectively to avidin, so that the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten and one of the various types of antibodies mentioned above is conjugated with an anti-hapten antibody. Thus, indirect conjugation of the label with the antibody can be achieved.

In addition to the sample preparation procedures discussed above, additional treatment of tissue sections may be required before, during or after IHC. For example, epitope repair methods, such as heating tissue samples in citrate buffer, can be performed (eg, Leong et al., Appl. Immunohistochem. 4 (3): 201 (1996)). Reference).

After any blocking step, the tissue sections are exposed to the primary antibody for a sufficient time under appropriate conditions to allow the primary antibody to bind to the target protein antigen in the tissue sample. Conditions suitable for achieving this can be determined by routine experimentation. The extent to which the antibody binds to the sample is determined using any one of the detectable labels discussed above. Preferably, the label is an enzyme label (eg HRPO) that catalyzes the chemical modification of a chromogenic substrate, such as a 3,3'-diaminobenzidine chromogen. Preferably, the enzyme label is conjugated to an antibody that specifically binds to a primary antibody (eg, the primary antibody is a rabbit polyclonal antibody and the secondary antibody is a goat anti-rabbit antibody).

Optionally, the antibody used in IHC analysis to detect expression of GalNac-T14 is an anti-GalNac-T14 antibody. Alternatively, antibodies to other GalNac-T antigens cross-reactive with GalNac-T14 can be used. Optionally, the anti-GalNac-T14 antibody is a monoclonal antibody.

The specimen thus prepared may be mounted to cover the cover. The slide evaluation can then be determined, for example using a microscope, and the staining intensity criteria routinely used in the art can be used. Dye intensity criteria can be evaluated as follows.

Dyed pattern score No staining observed in cells 0 Faint / nearly undetectable staining detected in more than 10% of cells 1+ Mild to moderate staining is observed in more than 10% of cells 2+ Moderate to strong staining observed in more than 10% of cells 3+

Typically, a staining pattern score of at least about 2+ in such IHC assays is believed to predict or indicate the sensitivity of mammalian cells (eg, mammalian cancer cells) to Apo2L / TRAIL or death receptor agonist antibodies.

In an alternative method, the complex can be detected after contacting the sample with an antibody specific for the biomarker under conditions sufficient to form an antibody-biomarker complex. The presence of biomarkers can be detected in a number of ways, such as by Western blotting and ELISA procedures for analyzing a wide variety of tissues and samples, including plasma and serum. A wide range of immunoassay techniques using this assay format are available and see, for example, US Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. Such methods include traditional competitive binding assays as well as non-competitive types of single site and double site or “sandwich” assays. Such assays also include direct binding of the labeled antibody to the target biomarker.

Sandwich assays are the most useful and commonly used assays. There are many variations of the sandwich assay technique, all of which are intended to be included in the present invention. Briefly, in a typical forward assay, an unlabeled antibody is immobilized on a solid substrate and the sample to be tested is contacted with bound molecules. After an appropriate incubation period, a secondary antibody specific for the antigen, labeled with a reporter molecule capable of generating a detectable signal, is added for a time sufficient to allow the antibody-antigen complex to form, and the antibody-antigen- Incubate for a time sufficient to allow another complex of labeled antibodies to form. Any unreacted material is washed away and the presence of the antigen is determined by observing the signal generated by the reporter molecule. The results can be qualitative by simple observation of the visible signal or can be quantified by comparison with a control sample containing a known amount of biomarker.

Modifications of the anterior assay include simultaneous assays in which both the sample and the labeled antibody are added simultaneously to the bound antibody. Such techniques are well known to those skilled in the art, including any small variations that will be readily apparent. In typical anterior sandwich assays, primary antibodies with specificity for biomarkers are covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer and the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid support may be in the form of tubes, beads, disks of microplates, or any other surface suitable for conducting an immunoassay. The binding process is well known in the art and generally consists of crosslinking, covalent bonding or physical adsorption, and the polymer-antibody complexes are washed at the time of preparation of the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and subjected to suitable conditions (eg room temperature to 40 ° C., such as 25 ° C. to 32 ° C.) for a sufficient time (eg 2-40 minutes or overnight if more convenient). Incubated under C) to allow any subunits present in the antibody to bind. After the incubation period, the antibody subunit solid phase is washed, dried and incubated with secondary antibodies specific for a portion of the biomarker. The secondary antibody is linked to a reporter molecule that is used to direct the binding of the secondary antibody to the molecular marker.

An alternative method involves immobilizing a target biomarker in a sample and then exposing the immobilized target to a specific antibody that may or may not be labeled with a reporter molecule. Depending on the amount of target and the intensity of the reporter molecule signal, the bound target can be detected by direct labeling with the antibody. Alternatively, a labeled secondary antibody specific for the primary antibody is exposed to the target primary antibody complex to form a ternary complex of the target primary antibody secondary antibody. The complex is detected by the signal emitted by the reporter molecule. As used herein, the term “reporter molecule” refers to a molecule that provides, by chemical properties of the molecule, an analytically identifiable signal that allows detection of antigen-bound antibodies. The most commonly used reporter molecules in this type of assay are enzymes, fluorophores or radionuclide containing molecules (ie radioisotopes) and chemiluminescent molecules.

For enzyme immunoassays, enzymes are generally conjugated to secondary antibodies by glutaraldehyde or periodate. However, as will be readily appreciated, there are a wide variety of bonding techniques readily available to those skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, galactosidase and alkaline phosphatase. Substrates used with specific enzymes are generally selected such that detectable color changes are produced upon hydrolysis by the corresponding enzyme. Examples of suitable enzymes include alkaline phosphatase and peroxidase. Fluorescent substrates from which fluorescent products are produced can also be used rather than the chromogenic substrates mentioned above. In all cases, the enzyme labeled antibody is added to the primary antibody-molecular marker complex to bind, followed by washing off excess reagent. Then, a solution containing a suitable substrate is added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the secondary antibody to provide a qualitative visible signal, which can generally be further quantified spectrophotometrically to provide an indication of the amount of biomarker present in the sample. Alternatively, fluorescent compounds such as fluorescein and rhodamine can be chemically coupled to the antibody without altering the binding ability of the antibody. When activated by irradiation of light of a particular wavelength, a fluorochrome-labeled antibody absorbs light energy to induce the molecule into an excitable state and then emits light in a characteristic color that is visually detectable with an optical microscope do. As in the EIA, the fluorescently labeled antibody will bind to the primary antibody-molecular marker complex. After removal of the unbound reagent by washing, the remaining ternary complex is exposed to light of the appropriate wavelength and the fluorescence observed indicates the presence of the molecular marker in question. Both immunofluorescence and EIA techniques are well established in the art. However, other reporter molecules may also be used, such as radioisotopes, chemiluminescent or bioluminescent molecules.

It is contemplated that the techniques described above can also be used to detect expression of GalNac-T14.

The methods of the present invention further comprise a protocol for examining the presence and / or expression of GalNac-T14 mRNA in tissue or cell samples. Methods of evaluating mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization, Northern blots and related techniques using labeled GalNac-T14 riboprobes) and various nucleic acid amplification assays. (Eg, RT-PCR using complementary primers specific for GalNac-T14, and other methods of detection of amplification such as branched DNA, SISBA, TMA, etc.).

Mammalian tissue or cell samples can be conveniently analyzed for, for example, GalNac-T14 mRNA using Northern, dot blot or PCR analysis. For example, RT-PCR assays such as quantitative PCR assays are well known in the art. In an exemplary embodiment of the invention, a method of detecting GalNac-T14 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using one or more primers; Amplifying the cDNA thus produced using GalNac-T14 polynucleotides as sense and antisense primers to amplify the internal GalNac-T14 cDNA; And detecting the presence of the amplified GalNac-T14 cDNA. In addition, such methods may include one or more steps that allow to determine the level of GalNac-T14 mRNA in a biological sample (eg, comparative control of "housekeeping" genes, such as actin family members). by simultaneously examining the levels of mRNA sequences). Optionally, the sequence of the amplified GalNac-T14 cDNA can be determined.

Material embodiments of this aspect of the invention may optionally be directed to GalNac-T14 primers and primer pairs, and nucleic acid molecules of the invention or any portion thereof, such that the polynucleotide of the invention or any particular portion thereof is specifically amplified. Or probes that hybridize specifically. The probe may be labeled with a detectable marker such as a radioisotope, fluorescent compound, bioluminescent compound, chemiluminescent compound, metal chelator or enzyme. Such probes and primers can be used to detect the presence of GalNac-T14 polynucleotides in a sample and as a means for detecting cells expressing GalNac-T14 protein. As will be appreciated by those skilled in the art, a large number of different primers and probes can be prepared based on the sequences provided herein and used effectively for the amplification, cloning and / or determination of the presence and / or levels of GalNac-T14 mRNA.

Any method of the present invention includes a protocol for investigating or detecting mRNA, such as GalNac-T14 mRNA, in a tissue or cell sample by microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probe is then hybridized to the nucleic acid array immobilized on a solid support. The array is formed to know the sequence and position of each member of the array. For example, a collection of genes that are likely to be expressed in certain disease states can be arranged on a solid support. Hybridization of labeled probes with specific array members indicates that the sample from which the probe is derived expresses the gene. Differential gene expression analysis of diseased tissue can provide useful information. Microarray technology uses nucleic acid hybridization technology and computer technology to evaluate mRNA expression profiles of thousands of genes in a single experiment (see, eg, WO 01/75166 (October 11, 2001); array construction For discussion, see, eg, US Pat. No. 5,700,637, US Pat. No. 5,445,934 and US Pat. No. 5,807,522, Lockart, Nature Biotechnology, 14: 1675-1680 (1996); Cheung, VG et al., Nature Genetics 21 (Suppl) : 15-19 (1999). DNA microarrays are arrays of thumbnails that contain gene fragments that are synthesized directly on, or spotted on, a glass or other substrate. Thousands of genes are generally presented in a single array. A typical microarray experiment involves the following steps: 1. Preparation of a fluorescently labeled target from RNA isolated from a sample, 2. Hybridization of the labeled target to a microarray, 3. Washing, staining and scanning of the array, 4. Analysis of the scanned image and 5. Generation of gene expression profiles. Two main types of DNA microarrays are currently used: gene expression arrays containing oligonucleotide (usually 25-70mer) arrays, and PCR products made from cDNA. In forming the array, the oligonucleotides can be prefabricated and spotted on the surface or synthesized directly on the surface (in situ).

The Affymetrix GeneChip® system is a commercially available microarray system that includes an array made by direct synthesis of oligonucleotides on a glass surface. Probe / Gene Arrays: Oligonucleotides (usually 25-mers) are synthesized directly on glass wafers by a combination of semiconductor-based photolithography and solid phase chemical synthesis techniques. Each array contains up to 400,000 different oligomers, and each oligomer is present in millions of copies. Since oligonucleotide probes are synthesized at known locations on the array, hybridization patterns and signal intensities can be interpreted in terms of gene identity and relative expression levels by Affymetrix Microarray Suite software. Each gene is presented on an array by a series of different oligonucleotide probes. Each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide. A perfect match probe has a sequence that is exactly complementary to a particular gene and thus measures the expression of the gene. Mismatch probes differ from perfect match probes by one base substitution at a central base position, thus interfering with binding of the target gene transcript. This helps to determine the background and non-specific hybridization that contributes to the signal measured for the perfect match oligo. The microarray suite software subtracts the hybridization intensity of the mismatch probe from the hybridization intensity of the perfect match probe to determine the absolute or specific intensity value for each probe cell. Probes are selected based on information from Genbank and other nucleotide deposit institutions. This sequence is believed to recognize a unique region of the 3 'end of the gene. Hybridization of up to 64 arrays is performed at a time using a Ginchip hybridization oven ("rotary" oven). Fluidics stations perform washing and staining of probe arrays. It is fully automated and contains four modules, each holding one probe array. Each module is controlled independently through the Microarray Suite software using preprogrammed Fluidic protocol. The scanner is a confocal laser fluorescence scanner that measures the fluorescence intensity emitted by labeled cRNA bound to a probe array. A computer workstation with microarray suite software controls the fluidic station and scanner. The microarray suite software can control up to eight fluidic stations using preprogrammed hybridization, wash, and stain protocols for the probe array. The microarray suite software also uses an appropriate algorithm to obtain hybridization intensity data and convert it to the presence / absence call for each gene. Finally, the microarray suite software detects changes in gene expression between experiments by comparative analysis and formats the results into a .txt file, which can be used in other software programs for further data analysis.

Fluorescent in situ hybridization (FISH) assays can also be used to detect expression of biomarker mRNA using labeled probes. Such assays are known in the art (see, eg, Kallioniemi et al., 1992; US Pat. No. 6,358,682).

Expression of selected biomarkers can also be assessed by examining gene deletion or gene amplification. Gene deletion or amplification can be accomplished by any of a wide range of protocols known in the art, eg, conventional Southern blotting, Northern blotting to quantify transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA 77: 5201-5205 (1980)), dot blotting (DNA analysis), or in situ hybridization (eg FISH), cytogenetic methods, or suitably labeled probes using suitably labeled probes. It can be measured by comparative genomic hybridization (CGH) used. By way of example, these methods can be used to detect deletion of GalNac-T14 gene amplification.

Additionally, the methylation status of biomarkers, such as the GalNac-T14 gene, can be examined in tissue or cell samples. Abnormal demethylation and / or hypermethylation of CpG islands in the gene 5 ′ regulatory region often occurs in endless growth and transgenic cells, and as a result the expression of various genes may be modified. Various assays for investigating the methylation status of genes are well known in the art. For example, in the Southern hybridization approach, methylation-sensitive restriction enzymes that cannot cleave sequences containing methylated CpG sites can be used to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all CpG sites present on the CpG island of a given gene. This procedure involves initial modification of DNA with sodium bisulfite (converts all unmethylated cytosine to uracil) followed by amplification with primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference are also described, for example, in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al., Eds., 1995; De Marzo et al., Am. J. Pathol. 155 (6): 1985-1992 (1999); Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998, 7: 531-536; And Lethe et al., Int. J. Cancer 76 (6): 903-908 (1998).

Expression of GalNac-T14 in tissue or cell samples can also be examined by functional or activity-based assays. For example, assays known in the art can be used to determine or detect the presence of certain enzymatic N-acetylgalactosaminiltransferase activity in a tissue or cell sample (eg, Bennett et. al., J. Biol. Chem., 271: 17006-17012 (1996); Wang et al., BBRC, 300: 738-744 (2003); Hang et al., supra. available May 2005 at sciencedirect.com).

In the methods of the invention, it is contemplated that expression of Apo2L / TRAIL or receptor in a sample that binds a tissue or cell sample to Apo2L / TRAIL or a death receptor antibody may also be investigated. As described above and in the art, Apo2L / TRAIL is currently believed to bind to at least five different receptors, DR4, DR5, DcR1, DcR2 and OPG. Expression of Apo2L / TRAIL, DR4, DR5, DcR1, DcR2 and / or OPG can be detected at mRNA and protein levels using methods known in the art, including those described herein. By way of example, the IHC technique described above can be used to detect the presence of one of a number of such molecules in a sample. In methods where tissues or samples are examined for the presence of GalNac-T14 markers as well as for example for the presence of DR4, DR5 or DcR1, separate slides are prepared from the same tissue or sample, and each slide is prepared for each specific bio It is contemplated to be able to test with reagents specific for the marker or receptor. Alternatively, a single slide can be prepared from a tissue or cell sample and the antibodies directed for each biomarker or receptor can be used in conjunction with a multicolor staining protocol to allow individual biomarkers or receptors to be visualized and detected. Can be.

After it is determined that the tissue or cell sample expresses GalNac-T14, indicating that the tissue or cell sample is sensitive to Apo2L / TRAIL or death receptor antibody, an effective amount of Apo2L / TRAIL or death receptor antibody is administered to the mammal, It is contemplated to be able to treat disorders such as cancer or immune related disorders that afflict mammals. Diagnosis in mammals of the different pathological conditions described herein can be made by a skilled practitioner. Diagnostic techniques are available in the art, for example, to diagnose or detect cancer or immune related diseases in mammals. Cancer can be identified through techniques including, but not limited to, palpation, blood analysis, x-rays, NMR, and the like. Immune-related diseases are also easily identified.

Apo2L / TRAIL or death receptor antibodies may be administered according to known methods, such as intravenous administration as bolus or continuous infusion over time, intramuscular, intraperitoneal, intrathecal, subcutaneous, intraarticular, intravaginal, intramedullary, oral It may be administered by topical or inhalation routes. Optionally, administration can be performed via mini pump infusion using various commercially available instruments.

Effective dosages and schedules for the administration of Apo2L / TRAIL or death receptor antibodies can be determined empirically, and making such determinations is within the skill of the art. Single or multiple doses may be used. It is presently believed that when used alone, an effective dosage or effective amount of Apo2L / TRAIL may range from about 1 μg / kg body weight / day to about 100 μg / kg body weight / day or more. Interspecies variation in dosage is described, for example, in Mordenti et al., Pharmaceut. Res., 8: 1351 (1991), in a manner known in the art.

When in vivo administration of Apo2L / TRAIL is used, the normal dosage is, depending on the route of administration, from about 10 ng / mammal weight kg / day to 100 mg / mammal weight kg / day or more, preferably about 1 μg / It can vary from kg / day to 10 mg / kg / day. Guidance as to particular dosages and methods of delivery is provided in the literature; See, for example, US Pat. No. 4,657,760; 5,206,344; Or 5,225,212. Different formulations will be effective against different therapeutic compounds and different disorders, for example administration targeted to one organ or tissue is expected to require delivery in a different way than another organ or tissue.

It is contemplated that additional therapies may be used in the method. One or more other therapies include radiotherapy, cytokine (s), growth inhibitor (s), chemotherapeutic agent (s), cytotoxic agent (s), tyrosine kinases known in the art and defined in further detail above. Administration of inhibitors, ras farnesyl transferase inhibitors, angiogenesis inhibitors, and cyclin-dependent kinase inhibitors. It is contemplated that such another therapy may be used as a separate agent from Apo2L / TRAIL or death receptor antibodies. In addition, therapies based on therapeutic antibodies targeting tumor antigens such as Rituxan ™ or Herceptin ™, as well as anti-angiogenic antibodies such as anti-VEGF can be used.

Preparation and dosing schedules for chemotherapeutic agents can be used according to the manufacturer's instructions or as determined empirically by an experienced physician. Preparation and administration of such chemotherapeutic agents is described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may be administered before or after administration of Apo2L / TRAIL or a death receptor antibody, or may be administered concurrently.

Antibodies to other antigens such as CD20, CD11a, CD18, CD40, ErbB2, EGFR, ErbB3, ErbB4, Vascular Endothelial Factor (VEGF), or other TNFR family members (eg OPG, TNFR1, TNFR2, GITR, Apo-3, TACI) It may also be desirable to administer an antibody that binds to BCMA, BR3). Alternatively, or in addition, two or more antibodies that bind to the same or two or more different antigens disclosed herein may be administered to the patient jointly. Sometimes, it may be beneficial to also administer one or more cytokines to the patient. After administration, treated cells in vitro can be analyzed. For in vivo treatments, the treated mammals can be monitored in a variety of ways well known to the skilled physician. For example, tumor cells can be examined pathologically to analyze necrosis, or serum can be analyzed for immune system response.

For use in the above described or proposed uses, kits or articles of manufacture are also provided by the present invention. Such a kit may comprise a conveying means which is compartmentalized to receive one or more container means, such as vials, tubes, etc. in close proximity, each container means comprising one of the distinct elements used in the method. For example, one of the container means may comprise a detectably labeled or labeled probe. Such probes may be antibodies or polynucleotides specific for the GalNac-T14 protein or GalNac-T14 gene or message, respectively. If the kit uses nucleic acid hybridization to detect a target nucleic acid, the kit may be a container containing a nucleotide (s) for amplification of the target nucleic acid sequence and / or a reporter molecule, such as an enzyme, fluorescent or radioisotope label. -May also comprise a container containing a biotin-binding protein such as avidin or streptavidin.

The kits of the present invention typically comprise one or more other containers including the containers described above, and buffer inserts with diluents, filters, needles, syringes, and instructions for use, from a commercial and user standpoint. Will include as. The label may be present on the container to indicate that the composition is to be used for a particular therapeutic or non-therapeutic use, and may also indicate instructions for in vivo or in vitro use, such as those described above.

There are a number of embodiments in the kits of the invention. Typical embodiments provide instructions for using a GalNac-T14 antibody to assess the presence of a GalNac-T14 protein in a container, a label on the container, a composition contained within the container, and one or more types of mammalian cells. A kit comprising; Wherein the composition comprises a primary antibody that binds to a GalNac-T14 polypeptide sequence, wherein the label on the container indicates that the composition can be used to assess the presence of GalNac-T14 protein in one or more types of mammalian cells. The kit may further comprise a set of instructions and materials for preparing a tissue sample and applying the antibody and probe to the same section of the tissue sample. The kit may comprise both primary and secondary antibodies, wherein the secondary antibody is conjugated to a label, eg an enzyme label.

Another embodiment uses a GalNac-T14 polynucleotide to assess the presence of a GalNac-T14 RNA or DNA in a container, a label on the container, and a composition contained within the container, and one or more types of mammalian cells. A kit containing instructions for the; Wherein the composition comprises a polynucleotide that hybridizes to the complement of the GalNac-T14 polynucleotide under stringent conditions, and the label on the container can be used for the composition to assess the presence of GalNac-T14 in one or more types of mammalian cells. Indicates that there is.

Any other component within the kit may include one or more buffers (eg, blocking buffers, wash buffers, substrate buffers, etc.), other reagents, such as substrates (eg, chromophores) that are chemically modified by enzyme labels, epitopes. Repair solutions, control samples (positive and / or negative controls), control slide (s), and the like.

Various aspects of the invention are further described and described by the following examples, which are not intended to limit the scope of the invention.

Method and material:

Cell cultures and cell lines.

The following human cell lines: non-small cell lung cancer (NSCLC) cell lines: H2122, A427, H647, SK-MES-1, H838, H358, H2126, H460, H1703, H2405, H650, H1568, H1666, H322T, SW1573, H292, H1650, H522, EKVX, H661, H23, LXFL 529, H226, A549, H1781, H1299, HOP 62, H2009, HOP 92, H1793, H1975, H1651, calu-1, H1435, HOP 18, H520, H441, H2030, H1155, H1838, H596, HLFa; Pancreatic cancer cell lines: Panc 05.04, BxPC3, HPAC, SU.86.86, HuP-T3, PSN1, Panc 08.13, MiaPaCa-2, PA-TU-8988T, Panc 03.27, Capan-1, SW 1990, CFPAC-1, PA-TU -8902, Panc 02.03, Panc 04.03, PL45, Aspc-1, Hs766T, Panc 10.05, Panc1, Capan-2, HPAF-II, and NHL: JEKO-1, SU-DHL-4, OCI-LY-19, SR , Farage, DOHH-2, Toledo, WSU-NHL, KARPAS-422, GRANTA-519, Pfeiffer, HT, SC-1, DB. Cell lines were obtained from ATCC Depository (Manassa, Virginia), German Collection of Microorganisms and Cell Cultures (DSMZ), Japanese Cell Resources Bank (JCRB), or European Collection of Cell Cultures (ECACC), and 10% heat inactivated fetal bovine serum. Cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10 mM HEPES.

Cytotoxicity Assay.

Apo2L / TRAIL using the MTT assay (CellTiter 96® non-radioactive cell proliferation assay (Promega)), a colorimetric assay based on the ability of viable cells to reduce soluble yellow tetrazolium salt (MTT) to blue formazan crystals Or the amount of viable cells after treatment with the DR5 antibody. The MTT assay was performed by adding the premixed optimized dye solution to culture wells of 96-well plates containing various concentrations (0 to 1000 ng / ml) of Apo2L / TRAIL or DR5 antibody. During 4 hours incubation, the living cells convert the tetrazolium component of the dye solution to formazan product. The solubilization / stop solution was then added to the culture wells to solubilize the formazan product and the absorbance at 570 nm was recorded using a 96-well plate reader (SpectraMax). 570 nm absorbance readings are directly proportional to the number of cells commonly used in proliferation assays. The maximum absorbance for the formazan product is 570 nm and the pure solution appears blue, but the color at the end of the assay may not be blue and exists compared to other components in the culture medium (including serum, acidified phenol red and non-reducing MTT). According to the amount of Mazan.

Cell number was optimized by performing cell titration to generate an assay signal near the high end of the linear range of the assay. Since different cell types have different levels of metabolic activity, this was done separately for each cell line. For most tumor cells investigated, 5,000 cells / well to 20,000 cells / well were used.

The following is a step-by-step description of the analysis used:

1. Cells used for biological assays were taken from mother liquor culture.

2. Determine cell number and trypan blue viability and suspend the cells to a final number of 5,000 to 20,000 cells / well.

3. Dispense 50 μl of cell suspension into 96-well plates.

4. Plates were incubated overnight at 37 ° C. in a humidified 5% CO ₂ atmosphere.

5. 50 μl culture medium containing Apo2L / TRAIL or DR5 antibodies at various concentrations ranging from 0 to 1000 ng / ml was added to the samples in 96-well plates. Controls were 50 μl culture medium (no Apo2L / TRAIL or DR5 antibody) and 100 μl culture medium (no cells). Each experiment was performed on three independent days in three sets of wells. The total volume of the wells was 100 μl / well.

6. The plates were incubated at 37 ° C. for 72 hours in a humidified 5% CO ₂ atmosphere.

7. 15 μl of dye solution was added to each well.

8. Plates were incubated at 37 ° C. in a humidified 5% CO ₂ atmosphere for less than 4 hours.

9. 100 μl of solubilization / stop solution was added to each well.

10. Plates were incubated overnight at 37 ° C.

11. Absorbance at 570 nm wavelength was recorded using a 96-well plate reader. A reference wavelength of 750 nm was used to reduce the background caused by cell debris, fingerprints and other nonspecific absorbances.

12. The mean of the absorbance values for the negative control was used as the blank value and subtracted from all other readings. The amount of viable cells was calculated by dividing the average of absorbance values for each concentration of Apo2L / TRAIL or DR5 antibody by the average of the absorbance values of the positive control (100% viable cells-untreated).

13. The percentage of viable cells (Y axis) versus the concentration of Apo2L / TRAIL or DR5 antibody (X axis, logarithmic scale) was plotted and the IC 50 value was defined as the X axis value (ng / mL) corresponding to 50% viable cells. Determined by.

Affymetrix  Labeling Protocol

OD260 / 280 readings were taken for all samples and samples were run on the BioAnalyzer. 5 μg of high quality total RNA was used.

A. First Strand cDNA Synthesis:

1. Primer Hybridization

DEPC-H 2 O: x μl. Mixing by vortexing. Spin fast.

RNA (5 μg): y μl. Incubate at 70 ° C. for 10 minutes.

Spike (1: 4 dilution of mother liquor to 5 μg): 1 μl. Rapid Spin and Place on Ice

T7- (dT) 24 primer: 1 μl

Volume: 12 μl

2. Temperature adjustment

5 × -first strand cDNA buffer: 4 μl

7 μl of mix (to the left) is added to each sample.

0.1 M DTT: 2 μl. Vortex mixing. Spin fast.

10 mM dNTP mix: 1 μl. Incubate at 42 ° C. for 2 minutes.

Volume: 7 μl

3. First Strand Synthesis

1 μL SSII RT is added to each sample.

SSII RT: 1 μl. Mix by pipetting up or down or lightly vortexing.

Spin fast.

Total volume: 20 μl. Incubate at 42 ° C. for 1 hour.

B. Second Strand cDNA Synthesis

1. Place the first strand reactant on ice. Simply centrifuge to drop the concentrate on the side of the tube.

2. Prepare the following second strand master mix.

DEPC-treated H2O: 91 μl

5 × -second strand reaction buffer: 30 μl

10 mM dNTP mixture: 3 μl

10 U / μl DNA ligase: 1 μl

10 U / μl DNA Polymerase I: 4 μl

2 U / μl RNAse H: 1 μl

Total volume: 130 μl

3. Add 130 μl of the second strand master mix to 20 μl of the first strand cDNA. (Final volume = 150 μl)

4. Pipette up and down or lightly vortex to mix. Spin fast.

5. Incubate at 16 ° C. for 2 hours in a cooling bath.

6. Add 2 μl [10 U] T4 DNA polymerase. Pipette up and down or lightly vortex to mix. Spin fast.

7. Incubate at 16 ° C. for 5 minutes.

8. Add 10 μl 0.5 M EDTA. Lightly vortexed. Spin fast.

9. Proceed with cleaning procedure for cDNA or store at -20 ° C for later use.

Cleaning of Double-Strand cDNAs (Gin-Chip Sample Cleaning Module)

1. Add 600 μl cDNA binding buffer to the 162 μl final double stranded cDNA synthesis formulation. Mix for 3 seconds by vortexing.

2. Check the color of the mixture is yellow (similar to cDNA binding buffer without cDNA synthesis reaction). If the color of the mixture is orange or purple, 10 μl of 3 M sodium acetate (pH 5.0) is added and mixed. The color of the mixture will turn yellow.

3. Apply 500 μl of sample to a cDNA wash spin column placed in a 2 ml collection tube and centrifuge at ≧ 8,000 × g (≧ 10,000 rpm) for 1 minute. Dispose of flow-throught as * hazardous waste.

4. Reload the remaining mixture (262 μl) into the spin column and centrifuge as above. Dispose of the pass-through as * hazardous waste and discard the collection line.

5. Transfer the spin column to a new 2 ml collection tube (provided). 750 μl of cDNA wash buffer is pipetted onto the spin column. Centrifuge at ≧ 8,000 × g (≧ 10,000 rpm) for 1 minute. Discard the pass-through.

6. Open the cap of the spin column and centrifuge for 5 minutes at full speed (≦ 25,000 × g). The column is placed in a centrifuge each time using a second bucket. The cap is placed on the connecting bucket so that it is oriented in the opposite direction to the rotation, ie if the rotation is clockwise, the cap is oriented counterclockwise. This prevents the cap from being damaged. Discard the pass-through and collection tube.

7. Transfer the spin column to a 1.5 ml collection tube. 10 μl of cDNA elution buffer is pipetted directly onto the spin column membrane. Ensure that the cDNA elution buffer is distributed directly onto the membrane. Incubate at room temperature for 1 minute and elute by centrifugation at maximum speed (≦ 25,000 × g) for 1 minute.

Set up and run the IVT reaction

Enzo: Bioarray HighYield RNA Transcript Labeling Kit (Part No. 900182)

1. Use 10 μl of washed double stranded cDNA.

2. Prepare the following IVT master mixes:

Distilled or Deionized H2O: 12 μl

10 × HY reaction buffer: 4 μl

10 × biotin labeled ribonucleotides: 4 μl

10 × DTT: 4 μl

10 × RNAase inhibitor mixture: 4 μl

20 × T7 RNA polymerase: 2 μl

Total volume: 30 μl

3. Add 30 μl IVT master mix to 10 μl double strand cDNA. (Total volume = 40 μl)

4. Pipette up and down or lightly vortex to mix. Spin fast.

5. Immediately place the tube in a 37 ° C. water bath. Incubate for 5 hours.

6. If RNA is not purified immediately, store at -20 ℃.

Cleaning of Biotin-labeled cRNA (GinChip Sample Cleaning Module)

1. Add 60 μl of H 2 O to the IVT reaction and mix by vortexing for 3 seconds.

2. Add 350 μL of IVT cRNA binding buffer to the sample, vortex and mix for 3 seconds.

3. Add 250 μl of ethanol (96-100%) to the lysate, pipet and mix well. Do not centrifuge.

4. Sample (700 μl) is applied to an IVT cRNA wash spin column placed in a 2 ml collection tube. Centrifuge at ≧ 8,000 × g (≧ 10,000 rpm) for 15 seconds.

5. Pass the eluate once more through the column. Centrifuge at ≧ 8,000 × g (≧ 10,000 rpm) for 15 seconds. Discard the pass-through as a hazardous waste and discard the collection line.

6. Transfer the spin column to a new 2-ml collection tube (supplied).

7. Add 500 μl of IVT cRNA wash buffer and wash by centrifugation at ≧ 8,000 × g (≧ 10,000 rpm) for 15 seconds. Discard the pass-through.

8. Pipette 500 μl 80% (v / v) ethanol onto the spin column and centrifuge at ≧ 8,000 × g (≧ 10,000 rpm) for 15 seconds. Discard the pass-through.

9. Open the cap of the spin column and centrifuge for 5 minutes at full speed (≦ 25,000 × g). Discard the pass-through and collection tube.

10. Transfer the spin column to a new 1.5 ml collection tube.

11. Pipette 11 μl of RNAase free water directly onto the spin column membrane. Let stand for 1 minute. Eluate by centrifugation at maximum speed (≦ 25,000 × g) for 1 minute.

12. Pipette 10 μl of RNAase free water directly onto the spin column membrane. Let stand for 1 minute. Eluate by centrifugation at maximum speed (≦ 25,000 × g) for 1 minute.

Quantification of cRNA (IVT Product)

Spectrophotometric analysis is used to determine RNA yield. A conversion rate of 1 OD at 260 nm equals 40 μg / ml RNA is applied.

OD at 260 nm and 280 nm is checked to determine sample concentration and purity.

Maintain close to A260 / A280 ratio 2.0 for pure RNA (a ratio of 1.9 to 2.1 is allowed).

For quantification of cRNA when using total RNA as starting material, cRNA yields adjusted to reflect carryover of unlabeled total RNA should be calculated. Using estimates of 100% carryover, adjusted cRNA yields are determined using the following formula:

Adjusted cRNA yield = RNAm-(total RNAi) (y)

RNAm = amount of cRNA measured after IVT (μg)

Total RNAi = starting amount of total RNA (μg)

y = fraction of cDNA reactant used in IVT

Fragmentation of cRNA for Targeting Agents

For fragmentation, adjusted cRNA concentrations are used.

1. Add 2 μl of 5 × fragmentation buffer every 8 μl of RNA + H 2 O.

20 μg cRNA: 1 to 32 μl

5 × fragmentation buffer: 8 μl

RNAase free water: 40 μl

Total volume: 40 μl

2. Incubate at 94 ° C. for 30 minutes. Immediately, place on ice after incubation.

Hybridization Target Preparation:

1. 20 × Eukaryotic Hybridization Control and Oligo B2 are heated at 65 ° C. for 5 minutes. Affymetrix Eukchip Eukaryote Hybridization Control Kit, Part # 900362 (for 150 reactions)

2. Lightly vortex, spin and settle.

3. Master mix (assuming fragmented cRNA concentration of 0.5 μg / μl):

Standard array (μl) final concentration.

Fragmented cRNA 15 μg 30 0.05 μg / μl

Oligo B2 (3 nM) 5 50 pM

20 × control spike 15 1.5, 5, 25, 100 pM

(Bio B, C, D, Cre)

Herring Sperm DNA 3 0.1 mg / ml

Acetylated BSA 3 0.5 mg / ml

Hu cot-1 DNA (1 mg / ml) 30 0.1 mg / ml

2X MES Hyb Buffer 150 1 ×

H2O 64

Final volume 300

4. Aliquot 270 μl master mix into tubes and add 30 μl of fragmented cRNA to each tube. This is the hybridization mix.

5. Allow the probe array to equilibrate to room temperature immediately before use.

6. Charge the probe array with 1 × MES Hyb buffer and incubate at 45 ° C., 60 rpm for 10 minutes in a rotary oven.

7. Heat the hybridization mix in a 99 ° C. water bath for 5 minutes.

8. Transfer the hybridization mix to a 45 ° C. water bath for 5 minutes.

9. Centrifuge the hybridization mix at full speed for 5 minutes.

10. Remove 1 × MES Hyb buffer from the probe array.

11. Charge the upper 200 μl hybridization mix to the probe array.

12. Seal the diaphragm with Tough-Spots.

13. Hybridize the probe array at 45 ° C., 60 RPM for 19 hours.

14. Clean, stain and scan probe arrays according to the Affymetrix protocol.

Affymetrix  material

subject Seller catalogue # T7- (dT) 24 primer
Control spike
Superscript II / 5 × First Strand Buffer / 0.1 M DTT
5 × second strand buffer
10 mM dNTP
10 U / μl E. coli DNA ligase
10 U / μl E. coli DNA polymerase I
2 U / μL RNA Degradase H
10 U / μl T4 DNA Polymerase
0.5 M EDTA
ENZO High Yield RNA Transcript Labeling Kit
Gene Chip Sample Cleaning Module
Acetylated bovine serum albumin
Goat IgG-Reagent Grade
Anti-Streptavidin Antibody (Goat) (Biotinylated)
R-Phycoerythrin Streptavidin
20 × SSPE
Eukaryotic Control Kit
Water (molecular biology grade)
Human Cot-1 DNA
5 M NaCl (no RNAase, no DNAase)
Defoamer 0-30
10% Tween-20
MES free acid monohydrate
MES sodium salt
EDTA disodium salt (0.5 M solution)
Tough Spots, Label Dots
Gin Chip Hybridization Oven 640
Ginchip Scanner 3000 with Workstation
Hydrology station
Autoloader with External Barcode Reader Biosearch technologies
In-house
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Sigma
Affymetrix or ENZO
Affymetrix
Invitrogen
Sigma
Vector labs
Molecular probes
BioWhittaker
Affymetrix
Ambion
Roche
Ambion
Sigma
Pierce chemical
Sigma
Sigma
Sigma
USA Scientific
Affymetrix
Affymetrix
Affymetrix
Affymetrix goods ordered
-
18064-014
10812-014
18427-088
18052-019
18010-025
18021-071
18005-025
E-7889
900182 (ENZO)
900371
15561-020
I-5256
BA-0500
S-866
51214
900362
9934
1-581-074
9760
A-8082
28320
M5287
M3885
E7889
9902
800139
00-0074
00-0081
00-0129

Quantitative PCR

cDNA synthesis:

ingredient Volume (μl) 10 × RT buffer 10 25 × dNTP Mixture 4 10 × random primer 10 MultiScribe RT (50 U / μl) 5 H2O without RNAase 21 RNA (100 ng) 50 Final volume 100

Incubation Conditions :

25 ° for 10 minutes

37 ° for 2 hours

TaqMan reaction using ABI Prism 7700 sequencing detector:

ingredient Volume (μl) TaqMan Universal PCR Master Mix (2 ×) 25 TaqMan Probe (20 ×)
(Assays-on-Demand ™) 2.5 cDNA (100 ng) 2 H2O 20.5 Final volume 50

Thermal Cycling Conditions :

95 ° for 10 minutes

40 cycles: 95 ° for 15 seconds

           60 ° for 1 minute

TaqMan 프로브: Assays-on-Demand™ (TaqMan® MGB 프로브, FAM™ 염료-표지됨)

TaqMan Probe: Assays-on-Demand ™ (TaqMan® MGB probe, FAM ™ dye-labeled)

각각의 반응에 첨가된 샘플 RNA (cDNA)의 양을 표준화하기 위해 내인성 대조군 GAPDH (프로브 농도 100 nM, 전방향 및 역방향 프라이머 농도 200 nM)의 증폭을 수행하였다.

Amplification of the endogenous control GAPDH (probe concentration 100 nM, forward and reverse primer concentration 200 nM) was performed to normalize the amount of sample RNA (cDNA) added to each reaction.

Relative quantification was performed using standard curve methods. For standardization of quantification in endogenous controls, standard curves were prepared for both target and endogenous reference. For each experimental sample, the amount of target and endogenous reference was determined from the appropriate standard curve. The amount of target was then divided by the amount of endogenous reference to obtain normalized target values. One of the experimental samples served as a calibrator or 1 × sample. Each normalized target value was then divided by the calibration standardized target value, resulting in relative expression levels.

Experiment result:

Experiments were performed using the methods and materials described above. The results of this experiment are described in FIGS. 5-9 as discussed below.

5 shows Apo2L (+ 0.5% fetal bovine serum “FBS” or 10% FBS) or crosslinked “XL” or uncrosslinked DR5 monoclonal antibody “mab” (+0.5) as measured in the MTT cytotoxicity assay. IC50 Summary Plots of data obtained from analyzing non-small cell lung cancer (“NSCLC”) cell lines for sensitivity or resistance to apoptotic activity of% fetal bovine serum “FBS” or 10% FBS) are provided.

6 shows Apo2L (+ 0.5% fetal bovine serum “FBS” or 10% FBS) or crosslinked “XL” or uncrosslinked DR5 monoclonal antibody “mab” (+0.5) as measured in the MTT cytotoxicity assay. An IC50 summary chart of the data obtained in analyzing pancreatic cancer cell lines for sensitivity or resistance to apoptotic activity of% fetal bovine serum "FBS" or 10% FBS) is provided.

7 shows Apo2L (+ 10% fetal bovine serum “FBS”) or crosslinked “XL” or uncrosslinked DR5 monoclonal antibody “mab” (+ 0.5% fetal bovine serum), as measured in the MTT cytotoxicity assay. IC50 summary plots of data obtained from analyzing non-Hodgkin's lymphoma cancer (“NHL”) cell lines for sensitivity or resistance to apoptotic activity of “FBS” or 10% FBS) are provided.

8A and 8B are comparisons of sensitivity (“sen”) or resistance (“RES”) of selected NSCLC, pancreatic cancer, and NHL cell lines to DR5 antibodies, as measured by GalNac-T14 mRNA expression, and GalNac-T14 Provides a correlation for the expression of.

9A and 9B provide bar chart graphs of various NSCLC, pancreatic cancer, and NHL cell lines ranked in descending order by level of GalNac-T14 mRNA expression pattern.

Apoptosis Apoptosis programs play an important role in the development and starburst of multicellular organisms (Danial et al., Cell, 116: 205 (2004)). Intracellular stimuli can trigger apoptosis through cell-endogenous pathways, which rely on members of the Bcl-2 gene superfamily to activate apoptotic caspase devices (Cory et al., Nat Rev. Cancer, 2: 647 (2002)]. Certain cytokines belonging to the Tumor Necrosis Factor (TNF) superfamily can activate apoptosis via the cell-exogenous pathway by interacting with some receptors containing functional apoptosis-inducing "killing domains" (DD). (Ashkenazi et al., Science, 281: 1305 (1998)). Fas ligand (FasL) stimulates apoptosis via Fas (Apo1 / CD95), whereas Apo-2 ligand / TNF-related apoptosis-inducing ligand (Apo2L / TRAIL) is a DR4 (TRAIL-R1) and / or DR5 (TRAIL-R2) triggers apoptosis (LeBlanc et al., Cell Death Differ., 10:66 (2003)). Upon binding to cognate ligands, these receptors bind to the adapter molecule FADD (Fas associated death domain), which recruits apoptosis-initiator caspase-8 to form a death-induced signaling complex (DISC) (eg Kischkel et al., EMBO J., 14: 5579 (1995); Kischkel et al., Immunity, 12: 611 (2000). DISC-association stimulates caspase-8, and then caspase-8 cleaves and activates effector proteases such as caspase-3, 6, and 7, resulting in an apoptotic killing program. In many cell types, cross-talk to cell-endogenous pathways can further amplify cell-exogenous killing signals (Scaffidi et al., J. Biol. Chem., 274: 1541 (1999)]. Apo2L / TRAIL induces apoptosis in various tumor cell types with little or no effect on normal tissue, suggesting that Apo2L / TRAIL may be useful for cancer therapy (eg, Ashkenazi, Nat. Rev. Cancer, 2: 420 (2002); see Kelley et al., Curr. Opin. Pharmacol., 4: 333 (2004). Modifications in various components of the apoptosis pathway can reduce Apo2L / TRAIL sensitivity in certain cancer cell lines (Igney et al., Nat. Rev. Cancer, 2: 277 (2002)).

Various experiments were performed according to the methods and protocols described below, and data are provided in FIGS. 10-15.

To investigate the sensitivity to receptor activation, cell survival was tested as a function of Apo2L / TRAIL concentration in a panel of human cancer cell lines comprising 23 pancreatic adenocarcinomas, 18 malignant melanoma, and 36 colorectal adenocarcinomas (FIG. 10A and data not shown). This analysis classified 29/77 (38%) of the cell lines as highly or moderately sensitive to Apo2L / TRAIL. The Apo2L / TRAIL concentrations required to achieve 50% cell death in these 29 cell lines ranged from 3 to 800 ng / ml, with an average of 250 ng / ml.

Panels of cell lines were also examined by gene-expression profiling using a microarray of 54,613 gene-probe sets. Despite some prediction, mRNA levels of the O-glycosylation enzyme ppGalNAcT-14 were expressed in pancreatic cancer and melanoma cell lines that showed strong or moderate sensitivity to Apo2L / TRAIL compared to the corresponding resistant cell line ( p = 0.5 × 10 ⁻⁴ (Fisher's exact validation), cutoff values repeatedly assigned 750 for pancreatic carcinoma and 300 for melanoma) (FIG. 10B). Most Apo2L / TRAIL-sensitive colorectal cancer cell lines showed high mRNA expression of the related O-glycosylation enzyme ppGalNAcT-3, but several resistant cell lines also expressed these genes, resulting in weaker but significant differences (p = Cutoff value assigned to 0.026, 2000) (FIG. 10C, bottom). Exceptions in the overall panel were as follows: (a) 5/29 (17%) cell lines expressing ppGalNAcT-14 or ppGalNAcT-3 that are sensitive but below cutoff levels; (b) 16/48 (33%) resistant cell line whose ppGalNAcT-14 or ppGalNAcT-3 levels exceeded cuts despite being resistant. By examining mRNA expression of other O-glycosylase enzymes in colorectal cell lines, higher levels of Fut-6 were found in 10/12 (83%) of sensitive cells compared to 6/24 (25%) of resistant cell lines. Detected (p = 0.013; cutoff value assigned to 200) (FIG. 10C, top). Combined expression of ppGalNAcT-14 in pancreatic cancer and melanoma and Fut-6 in colorectal cancer cell lines correlated very strongly with Apo2L / TRAIL sensitivity (p = 1.83 × 10 ⁻⁷ , N = 77). These gene-sets accurately predicted sensitivity or resistance to 23/32 (72%) of marker-positive cell lines and 39/45 (87%) of marker-negative cell lines, respectively.

Apo2L / TRAIL sensitivity was also investigated in vivo using tumor xenografts. Treatment of mice with tumors derived from the Fut-6-positive colorectal cancer cell lines Colo205 and DLD-1 with Apo2L / TRAIL for 5 days resulted in tumor regression, which then significantly delayed tumor progression (FIG. 10D). In contrast, tumors derived from the Fut-6-negative colorectal cancer cell lines Colo320 and RKO did not respond to this treatment.

ppGalNAcT-3 / Fut-6-positive Colo205 cell line was preincubated with pan O-glycosyl transferase inhibitor benzyl-GalNAc (Delannoy et al., Glycoconj., 13: 717 (1996)) to Apo2L / TRAIL Sensitivity to was significantly reduced (FIG. 11A), suggesting a functional association between O-glycosylation and Apo2L / TRAIL signaling. To further investigate this, specific small interfering (si) RNA oligonucleotides targeting ppGalNacT-14, ppGalNacT-3, or Fut-6 mRNAs were used. To exclude off-target effects, multiple non-overlapping multiple siRNAs were synthesized for each gene and the ability to reduce target expression by quantitative RT-PCR was identified (FIG. 14A). siRNA specificity was further confirmed with the mutant ppGalNacT-14 plasmid containing six 'silent' nucleotide changes in the siRNA-target region (Editorial, Nat. Cell. Biol., 5: 489 (2003)). 14B). Transfection of the ppGalNAcT-14-positive PSN-1 pancreatic carcinoma and Hs294T melanoma cell line with ppGalNAcT-14 siRNA substantially reduced the sensitivity to Apo2L / TRAIL, whereas caspase-8 siRNA, as expected, had essentially complete protection. Provided (FIG. 11B). Similarly, transfection of DLD-1 or C170 colorectal cancer cells with GalNAcT-3 or Fut-6 siRNA significantly reduced the sensitivity to Apo2L / TRAIL (FIGS. 11C and 14C). In summary, GalNAcT-14 siRNA reduced sensitivity to Apo2L / TRAIL in 4/5 pancreatic cancer and 2/2 melanoma cell lines, while each ppGalNAcT-3 or Fut-6 siRNA was 2/3 colorectal cancer. Sensitivity was reduced in the cell line. In contrast, transfection of PSN-1 or Hs294T cells with GalNAcT-14 siRNA did not alter the sensitivity to the topoisomerase II inhibitor etoposide (FIG. 14D). Similarly, transfection of PSN-1 or C170 cells with GalNAcT-14 or GalNAcT-3 siRNA did not affect the sensitivity to a broad spectrum of protein kinase inhibitor staurosporin (FIG. 14E). Since both etoposide and staurosporin stimulate apoptosis via cell-endogenous pathways (Wei et al., Science, 292: 727 (2001)), these studies suggest that O-glycosylation enzymes It has been suggested that apoptosis signaling can be modulated through exogenous pathways.

Transfection of HEK293 cells with ppGalNAcT-14 showed cell death when co-transfected with DR4 or DR5, but not when co-transfected with the associated receptor Fas and TNFR1 or the cell-intrinsic pathway agonist Bax ( 11D). In addition, ppGalNAcT-14 transfection increased Apo2L / TRAIL sensitivity of resistant cell lines H1568 melanoma (FIG. 11E) and PA-TU-8902 and PL-45 pancreatic carcinoma (FIG. 14F), but the sensitivity to etoposide changed. (Data not shown). Overall, GalNAcT-14 overexpression sensitized 4/7 cell lines to Apo2L / TRAIL.

The effect of siRNA knockdown of ppGalNacT-14 or Fut-6 was investigated for Apo2L / TRAIL-induced caspase processing. In PSN-1 and DLD-1 cells transfected with control siRNA, Apo2L / TRAIL essentially induced complete caspase-8 processing leading to cleavage of Bid, caspase-9 and caspase-3 (FIG. 12A ). Transfection with caspase-8 siRNA prevented this event. PpGalNAcT-14 knockdown in PSN-1 cells or Fut-6 knockdown in DLD-1 cells, also Apo2L / TRAIL-induced processing of caspase-8, Bid, caspase-9, and caspase-3 (FIG. 12A) , And stimulation of caspase-3 / 7 activity (FIG. 12B) was significantly weakened. Apo2L / TRAIL-resistant RKO and SW1417 colorectal cancer cell lines expressing low levels of ppGalNAcT-3 and Fut-6 showed similar blockade at the caspase-8 processing level (FIG. 15A). Thus, O-glycosylase can modulate the Apo2L / TRAIL pathway upstream of events leading to caspase-8 activation.

Caspase-8 activation requires DISC assembly (Ashkenazi et al., Science, 281: 1305 (1998)). Analysis of Apo2L / TRAIL DISC (Kischkel et al., Immunity, 12: 611 (2000)) in PSN-1 and DLD-1 cells showed that knockdown of ppGalNAcT-14 or Fut-6 resulted in FADD and caspase Mobilization of -8, processing of DISC-binding caspase-8, and stimulation of DISC-associated caspase-8 enzyme activity was indicated (Sharp et al., J. Biol. Chem., 280: 19401 ( 2005)]) (FIGS. 12C, 12D, and 15B). ppGalNacT-14 or Fut-6 siRNA did not substantially change the amount of DR4 and DR5 in DISC, or the dose-dependent binding of Apo2L / TRAIL to PSN-1 or DLD-1 cells expressing both DR4 and DR5 ( 12C, 15B, and data not shown). Thus, ppGalNAcT-14 and Fut-6 do not appear to modulate apoptosis by affecting cell-surface receptor levels or Apo2L / TRAIL binding. In agreement, Apo2L / TRAIL sensitivity in a panel of 77 cell lines showed no significant correlation with cell-surface expression of cognate signaling receptors DR4 and DR5 or bait receptors DcR1 and DcR2 (data not shown). In addition, most siRNAs for ppGalNAcT-14, ppGalNacT-3, or Fut-6 did not alter DR4 and DR5 levels on PSN-1, C170, or DLD-1 cells (FIGS. 15C1 and 15C2). Both siRNAs did not result in a detectable reduction of DR4 and DR5 in certain cell lines (FIGS. 15C1 and 15C2). However, other siRNAs for the same enzyme inhibited Apo2L / TRAIL-induced apoptosis without altering receptor levels.

The extracellular domain (ECD) of human DR5 was expressed in Chinese hamster ovary cells and the secreted proteins were purified, acid hydrolyzed and the associated monosaccharides analyzed (FIG. 13A). N-linked glycans were not detected, consistent with the absence of predicted N-glycosylation sites in DR5 BCD. However, two samples from two independent experiments showed 3 moles of GalNAc and 3 moles of Gal per mole of DR5 ECD (FIG. 13A), which represented 3 on DR5 with core glycan GalNAc-Gal. A 0-linked modification of the dog site was suggested.

Protein O-glycosylation modifies serine or threonine. Using established bioinformatics tools to predict potential O-glycosylation sites (http://www.cbs.dtu.dk/services/NetOGlyc) (Julenius et al., Glycobiology, 15: 153 (2005))), two such regions within the common ECD sequence of the long (DR5-L) and short (DR5-S) splice variants of human DR5, and a third within the alternatively spliced region The site of was identified (FIG. 13B). The first amino acid segment (74-77) contains three serines; The second segment (130-144) contains five threonines, while the third segment has four threonines and three serines. The murine DR5 has a sequence similar to the first two fragments, each with two serines and four threonines, and the human DR4 also has two similar sequences containing one serine and five threonines. To test whether these sites are important for post-translational modifications of DR5, a set of DR5L and DR5S mutants were made and five threonines (DR5L-5T, DR5S-5T) of fragments 130-144, or these same five threonines In addition, three serines (DR5L-5T3S, DR5S-5T3S) of sections 74-77 were replaced with alanine. DR5 antibody immunoblot of lysates from HEK293 cells transfected with DR5L or DR5S showed the presence of expected DR5L or DR5S bands (FIG. 13C). The antibody also detected a DR5 band of higher molecular weight (MW), which was more abundant upon co-transfection of DR5L or DR5S with ppGalNAcT-14 compared to the control (FIG. 13C, asterisk). The abundance of these higher molecular weight bands and their augmentation with ppGalNAcT-14 were significantly reduced to DR5L-5T or DR5S-5T compared to wild type constructs, and were almost abolished with dDR5L-5T3S or DR5S-5T3S. These results suggested that higher molecular weight bands represent the O-glycosylated form of DR5: ppGalNAcT-14 promotes its formation, and the gradual removal by alanine substitution of the predicted O-glycosylation sites is such an effect. Back gradually. Transfection of HEK293 cells with murine DR5 or human DR4, DR5L, or DR5S showed cell death (FIG. 13D); Each DR5 mutant showed less activity than its corresponding wild type construct, and DR5S-5T3S (lacking all three sites) was the weakest. Co-transfection with ppGalNAcT-14 markedly enhanced cell death by all DR4 and DR5 constructs, except DR5S-5T3S, which showed significantly less activity.

Tumor samples from cancer or non-Hodgkin's lymphoma in the majority of normal-tissue and skin, lung, pancreas, breast, ovary, endometrium and bladder have cutoff values (500 for most cancers, 200 for skin cancers). Determined, ppGalNAcT-14 mRNA expression below 13E). However, a significant subset of tumor cells ranging from 10% of lobular breast cancer to 30% of lung cancer and diffuse large B-cell lymphoma showed overexpression of ppGalNAcT-14. Some cancer samples exceeded 1000 times the mRNA expression level of the corresponding normal tissue. Dynamic expression of ppGalNAcT-14 in cancer suggests that these genes, and possibly other related enzymes, can provide useful biomarkers for identifying tumors with greater sensitivity to Apo2L / TRAIL.

O-linked glycans exhibit a wide range of structural diversity and modulate various aspects of plasma membrane protein biology, including conformation, aggregation, passage, half-life, as well as cell adhesion and signaling activity (Hang et al., Bioorg). Med. Chem., 13: 5021 (2005); Hanisch, Biol. Chem., 382: 143 (2001). Cancer cells often show dramatic changes in the O-glycan profile, producing unique tumor-associated carbohydrate antigens (Brockhausen, Biochim. Biophys. Acta, 1473: 67 (1999); Dube et al., Nat. Rev. Drug Discovery, 4: 477 (2005); Fuster et al., Nat. Rev. Cancer, 5: 526 (2005)). O-glycosylation also plays an important role in the return of tumor cells to specific metastasis sites (Fuster et al., Cancer Res., 63: 2775 (2003); Ohyama et al., EMBO J., 18). : 1516 (1999); Takada et al., Cancer Res., 53: 354 (1993). Significant subsets of primary tumor samples from various human cancers, including colon and colorectal cell samples, melanoma cell samples, and chondroma cell samples, show elevated expression of the O-glycosylation enzyme ppGalNAcT-14.

Way

material.

Cell culture reagents were purchased from Gibco (Invitrogen / Gibco, Carlsbad, Calif.) And untagged soluble Apo2L / TRAIL was prepared as previously described (Lawrence et al., Nat. Med., 7: 383 (2001)], O-linked glycosylation inhibitor benzyl-a-GalNAc was purchased from Calbiochem, and all other chemicals (including etoposide and staurosporin) were purchased from Sigma Aldrich (St. Louis, MO). .

Cell culture and cell lines.

All 119 human carcinoma cell lines (see Supplemental Data for Name and Catalog #) were obtained from ATCC or DSMZ (Braunschweig, Germany) to provide 10% heat inactivated fetal bovine serum, 2 without antibiotics such as penicillin / streptomycin. Incubations were made at 37 ° C. and 5% CO ₂ in RPMI1640 supplemented with mM L-glutamine and 10 mM HEPES. 293 human embryonic kidney cells (catalog # CRL-1573) were also obtained from ATCC and cultured in 100% Dulbecco's Modified Eagle's Medium supplemented with 10% FBS. The ldlD CHO, an O-glycosylated mutant CHO cell line, was licensed from Dr. Monty Kreiger of MIT (Boston MA).

Cell viability assays and Apoptosis  Method.

To determine the IC 50 for Apo2L / TRAIL, cells were plated in triplicate in 96-well plates, attached for 24 hours and then treated with increasing concentrations of recombinant human Apo2L / TRAIL up to 1000 ng / ml. After 72 hours incubation, viability assay-MTT assay (Pierce) or CellTiter-Glo luminescent cell viability assay (Promega)-was applied according to the manufacturer's protocol. Each cell viability experiment was repeated three or more times in low concentration (0.5%) and high concentration (10% PBS) serum and defined moderately sensitive cell lines by variability between IC50s of independent experiments or between low and high concentration serum. . Cell lines were defined as sensitive based on induction of apoptosis of at least 50% of cells at an Apo2L / TRAIL concentration of 1 μg / ml and induced in independent experiments or in the presence of low (0.5%) to high (10%) serum. It was defined as moderately sensitive based on the variability of the amount of apoptosis. Apoptosis was quantified by flow cytometry analysis of the average percentage of harvested cells stained with Annexin V (BD Pharmingen) (attachment + suspension in medium).

Microarray Hybridization  And Data  analysis.

Total cell RNA was prepared from untreated cells (3 × 10 ⁶ ) using the RNeasy kit (Quiagen). As previously described (Hoffman et al., Nat. Rev. Genetics, 5: 229 (2004); Yauch et al., Clin. Cancer Res., 11: 8686 (2005)), labeled cRNA was prepared and hybridized to oligonucleotide microarrays (U133P GeneChip; Affymetrix Incorporated, Santa Clara, Calif.). Scanned image files were analyzed by GeneChip .3.1 (Affymetrix), Spotfire, GenePattern and Cluster / TreeView. To identify the genes that are most differentially expressed between the sensitive and resistant cell lines, the fold-change and absolute changes over the samples are tested and the ratio between the maximum and minimum values (maximum / minimum) and the difference between the maximum and minimum values. Gene-expression values were applied to a change filter that excludes genes with minimal change over the samples to be analyzed by comparing (maximum value-minimum value) with a predetermined value and excluding genes that do not follow both conditions.

Expression Constructs and Retroviral Transduction.

DNA fragments encoding ppGalNAcT-14 were cloned from cDNA pooled from Apo2L / TRAIL sensitive cell lines and inserted into the expression plasmid pcDNA3.1 (Invitrogen) with an N-terminal Flag tag. This construct is then applied to site directed mutagenesis (Quikchange mutagenesis kit, Stratagene) to siRNA silent mutations with 4-6 wobble base pair modifications in a sequence homologous to siRNA without altering the protein sequence. A sieve was produced. The mutation was spanned in the central 10 bp region of the 19 bp siRNA binding sequence. DNA sequences for DR5Long and DR5Short, DR4, murine TRAIL receptor, DR4, Fas (variant 1), TNFR1 and Bax (beta variant) were cloned from the cDNA pool and inserted into the pRK expression vector (Genentech). Site-directed mutation Mut4xTA (T130, T131, T132, T135) to four threonine alanine residues or Site-directed mutation Mut5xTA (T130, T131, T132, T135, T143) to alanine residues of four threonine ) Generated O-glycosylated mutants of DR5L and DR5S. Transient transfection into HEK293 cells with expression constructs of apoptosis-producing molecules was performed in 6-well plates at concentrations of 0.5 μg / well of apoptosis-inducing molecules and 2.0 μg of ppGalNAcT-14 or vector control. . Cells were transfected using Lipofectamine 2000 according to the manufacturer's protocol. After 48 hours incubation, cells were subjected to apoptosis assay.

To generate retroviral constructs, ppGalNAcT-14 and mutants were cloned into pQCXIP retroviral vector (Clontech). High titer retrovirus supernatants were generated using the ΦNX-Ampho helper cell line. Packaging cells were transfected with calcium phosphate (Invitrogen). The supernatant was isolated 48 hours after transfection, added to target cells with 10 μg / ml polybrene and centrifuged at 2700 rpm for 1 hour to enhance infection. After transduction, cells were selected with 2 μg / ml puromycin.

siRNA  Design and Transfection Protocols.

siRNAs for ppGalNAcT-14, ppGalNAcT-3, caspase-8 and DR5 were designed by Dharmacon (Lafayette, CO) using their proprietary selection criteria. The selected sequences were as follows:

siGalNAcT-14 (1): 5 'GACCATCCGCAGTGTATTA-dTdT 31 (= 14-4) (SEQ ID NO: 15)

siGalNAcT-14 (2): 5 'ATACAGATATGTTCGGTGA-dTdT 3' (= 14-6) (SEQ ID NO: 16)

siGalNAcT-3 (1): 5 'CCATAGATCTGAACACGTT-dTdT 3' (= 3-2) (SEQ ID NO: 17)

siGalNAcT-3 (2): 5 'GCAAGGATATTATACAGCA-dTdT 3' (= 3-7) (SEQ ID NO: 18)

siFut-6 (1) 5 'GUACCAGACACGCGGCAUA-dTdT 3' (= 6-1) (SEQ ID NO: 19)

siFut-6 (2) 5 'ACCGAGAGGUCAUGUACAA-dTdT 3' (= 6-2) (SEQ ID NO: 20)

si Caspase-8: 5 'GGACAAAGTTTACCAAATG-dTdT 3' (SEQ ID NO: 21)

siRNAs were purchased as double stranded RNA oligonucleotides and transfected into individual cell lines at a final concentration of 25 nM for each siRNA. RNA duplex (Dharmacon) for non-targeting sequences was used as a control. Cells were transfected using Lipofectamine2000 (Invitrogen) by reverse transfection where cells were added as suspension to the pre-plated lipid-siRNA complex. The concentration of Lipofectamine 2000 was according to the manufacturer's protocol. After 48 hours incubation, cells are harvested for mRNA analysis, or for an additional 24-72 hours with recombinant human Apo2L / TRAIL, etoposide or staurosporin for viability analysis, or 4 hours for western blot analysis Incubate for 8 hours or 24 hours.

Benzyl-a- GalNAc O- to Glycosylated  control.

Colo205 cells were grown for 72 hours in the presence of benzyl-a-GalNAc (2 mM or 4 mM). At this point, cells were plated back in 96-well plates and still attached for 24 hours in the presence of inhibitors. Cells were then stimulated with increasing concentrations of Apo2L / TRAIL as indicated and subjected to viability assay.

Quantitative PCR .

GalNacT-14 and GalNacT-3 transcript expression levels were assessed using standard Taqman techniques by quantitative RT-PCR. Transcript levels were normalized to the housekeeping gene, GAPDH, and the results are expressed as normalized expression values (= 2 ^{- DCt} ). Primer / probe sets for GalNacT-14 (Cat.

Immunoprecipitation, Weston Blot  Assay and Antibody.

IP: anti-Apo2L (2E11; ATCC accession number HB-12256), anti-DR4 (3G1 and 4G7, ATCC accession number PTA-99) and anti-DR5 (3H3, ATCC accession number 12534, and 5C7) monoclonal antibodies Was generated by Genentech, Inc. using the receptor-Fc fusion protein as antigen. Anti-DR4 (4G7) and anti-DR5 (5C7) monoclonal antibodies used to immunoprecipitate Apo2L / TRAIL DISC were prepared using the ImmunoPure Protein G IgG Plus Orientation Kit (Catalog # 44990, Pierce). ) To agarose. Anti-DR4 (3G1) and anti-DR5 (3H3) monoclonal antibodies used for immunodetection of DR4 / 5 in DISC immunoprecipitation were subjected to EZ-Link sulfo-NHS-LC biotinylation kit (Catalog # 21217, Pierce). Biotinylated. FLAG-tagged Apo2L / TRAIL was prepared and crosslinked with anti-FLAG antibody M2 (Sigma) as described (Kischkel, Immunity, 12: 611 (2000)). This experiment was performed as previously described for the Apo2L / TRAIL-FLAG + anti-FLAG DISC assay (Kischkel, supra). DR4 / 5 DISC immunoprecipitation experiments were also performed as described except that anti-DR4 (4G7) and anti-DR5 (5C7) monoclonal antibodies were directly conjugated to agarose for immunoprecipitation (Sharp et al., J. Biol. Chem., 280: 19401 (2005)].

WB: 5 × 10 ⁵ cells / well were seeded in 6 well plates. For RNAi knockdown experiments, cells were treated with several siRNAs for 48 hours, followed by Apo2L / TRAIL for 4 hours, 8 hours or 24 hours. After the indicated period of time, cells were washed in ice cold PBS and 1% Triton (20% HEPES pH 7.5, 10 mM KCL, 1.5 mM MgCl ₂ , 1 mM EDTA and 1 mM DTT) containing storage buffer ) Dissolved in X-100. 40 μg of protein for each sample was isolated under reducing conditions on a 10% or 10-20% gradient SDS-polyacrylamide gel. After transferring to nitrocellulose membranes (Schleicher & Schuell), the membranes were incubated in 10% fat-free milk powder for 1 hour and then incubated with the following primary antibodies for 1 hour: Goat anti-caspase-3 antibody (1 : 1000, R & D), rabbit anti-caspase-8 antibody (1: 1000, Pharmingen), mouse anti-caspase-9 antibody 5B4 (1: 1000, MBL), rabbit anti-Bid antibody (1: 1000, Pharmingen ), Rabbit anti-DR5 antibody (1: 500, Cayman) or goat anti-actin antibody (1: 200, Santa Cruz Biotechnology). Membranes were washed five times with TBS / 0.05% Tween and then incubated with affinity purified secondary antibodies (1: 5000, Biorad) conjugated to each peroxidase for 30 minutes. The membrane was washed again five times, developed using enhanced chemiluminescence (ECL, Amersham) and exposed to Kodak Biomax film.

Flow Cytometry / FACS analysis:

Surface expression of TNF family receptors DR4 and DR5 was determined by fluorescence-activated cell sorting (FACS) using a FACS Calibur flow cytometer (Becton Dickinson Immunocytometry System, San Jose, Calif.). C170 and PSN-1 cells transfected with the indicated siRNA for 48 hours were stained at 10 ° C. with primary antibody 4G7 (anti-DR4) or 3H3 (anti-DR5) or mouse IgG control antibody for 1 hour at 4 ° C. It was. Cells were then washed with PBS and then incubated with fluorescein (FITC) conjugated goat anti-mouse secondary antibody (Jackson Laboratories) for 30 minutes at 4 ° C. Cells were then analyzed using a FACS Calibur flow cytometer by flow cytometry.

Caspase  Method.

Caspase-3 / -7 activity was caspase buffer containing 100 μM of fluorescent peptide Ac-DEVD-AFC at 37 ° C. (50 mM HEPES pH 7.4, 100 mM NaCl, 10% sucrose, 1 mM EDTA, 0.1% CHAPS and 10 mM DTT) were analyzed in 40 μl. Molecular Devices fluorometer in kinetic mode with a 405-510 filter pair was used to continuously measure activity over the time indicated by the release of AFC from DEVD-AFC. For evaluation of caspase activity, 20 μg of whole cell protein (Triton X-100 extract) was used in 40 μl of caspase buffer (containing 100 μM DEVD-AFC).

CHO -origin DR5 Carbohydrate analysis.

The monosaccharide composition of CHO-cell-derived DR5 was obtained after hydrolysis with 4 N TFA. The released monosaccharides were analyzed on a Dionex BioLC HPLC system using high performance anion-exchange chromatography coupled to a pulsed ammeter detector.

Animal and Subcutaneous Xenograft Studies.

Female athymic nude mice (The Jackson Laboratory, Bar Harbor, ME, USA) were acclimated to an animal residence facility at Genentech for at least 1 week prior to experimental studies. All experimental procedures were approved by Genentech's Institutional Animal Care and Use Committee (IACAUC). Mice were inoculated subcutaneously with 5 × 10 ⁶ cells / mouse Colo205, DLD-1 and RKO or 20 × 10 ⁶ cells / mouse Colo320HSR human colon carcinoma cells (American Type Culture Collection). Tumor measurements were collected by digital calipers, and tumor volumes were calculated using the formulas □-□ (A = length) (B = width) ² . Once tumors reached a volume of about 150-200 mm 3, mice were randomized and vehicle or Apo2L / TRAIL (60 mg / kg / day) administered intraperitoneally on Days 0-4.

<110> Genentech, Inc. <120> ASSAYS AND METHODS USING BIOMARKERS <130> P2245R1 PCT <140> PCT / US2006 / 031785 <141> 2006-08-15 <150> US 60 / 708,677 <151> 2005-08-16 <150> US 60 / 808,076 <151> 2006-05-24 <160> 29 <210> 1 <211> 281 <212> PRT <213> Homo sapiens <400> 1  Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr    1 5 10 15  Cys Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys                   20 25 30  Val Ala Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met                   35 40 45  Gln Asp Lys Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu                   50 55 60  Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser                   65 70 75  Pro Cys Trp Gln Val Lys Trp Gln Leu Arg Gln Leu Val Arg Lys                   80 85 90  Met Ile Leu Arg Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu                   95 100 105  Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln                  110 115 120  Arg Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr                  125 130 135  Leu Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys                  140 145 150  Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser                  155 160 165  Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly                  170 175 180  Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu                  185 190 195  Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln Tyr Ile                  200 205 210  Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys Ser                  215 220 225  Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr                  230 235 240  Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg                  245 250 255  Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His                  260 265 270  Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly                  275 280 <210> 2 <211> 1042 <212> DNA <213> Homo sapiens <220> <221> Unsure <222> 447 <223> Unknown base <400> 2  tttcctcact gactataaaa gaatagagaa ggaagggctt cagtgaccgg 50  ctgcctggct gacttacagc agtcagactc tgacaggatc atggctatga 100  tggaggtcca ggggggaccc agcctgggac agacctgcgt gctgatcgtg 150  atcttcacag tgctcctgca gtctctctgt gtggctgtaa cttacgtgta 200  ctttaccaac gagctgaagc agatgcagga caagtactcc aaaagtggca 250  ttgcttgttt cttaaaagaa gatgacagtt attgggaccc caatgacgaa 300  gagagtatga acagcccctg ctggcaagtc aagtggcaac tccgtcagct 350  cgttagaaag atgattttga gaacctctga ggaaaccatt tctacagttc 400  aagaaaagca acaaaatatt tctcccctag tgagagaaag aggtccncag 450  agagtagcag ctcacataac tgggaccaga ggaagaagca acacattgtc 500  ttctccaaac tccaagaatg aaaaggctct gggccgcaaa ataaactcct 550  gggaatcatc aaggagtggg cattcattcc tgagcaactt gcacttgagg 600  aatggtgaac tggtcatcca tgaaaaaggg ttttactaca tctattccca 650  aacatacttt cgatttcagg aggaaataaa agaaaacaca aagaacgaca 700  aacaaatggt ccaatatatt tacaaataca caagttatcc tgaccctata 750  ttgttgatga aaagtgctag aaatagttgt tggtctaaag atgcagaata 800  tggactctat tccatctatc aagggggaat atttgagctt aaggaaaatg 850  acagaatttt tgtttctgta acaaatgagc acttgataga catggaccat 900  gaagccagtt ttttcggggc ctttttagtt ggctaactga cctggaaaga 950  aaaagcaata acctcaaagt gactattcag ttttcaggat gatacactat 1000  gaagatgttt caaaaaatct gaccaaaaca aacaaacaga aa 1042 <210> 3 <211> 468 <212> PRT <213> Homo sapiens <400> 3  Met Ala Pro Pro Pro Ala Arg Val His Leu Gly Ala Phe Leu Ala    1 5 10 15  Val Thr Pro Asn Pro Gly Ser Ala Ala Ser Gly Thr Glu Ala Ala                   20 25 30  Ala Ala Thr Pro Ser Lys Val Trp Gly Ser Ser Ala Gly Arg Ile                   35 40 45  Glu Pro Arg Gly Gly Gly Arg Gly Ala Leu Pro Thr Ser Met Gly                   50 55 60  Gln His Gly Pro Ser Ala Arg Ala Arg Ala Gly Arg Ala Pro Gly                   65 70 75  Pro Arg Pro Ala Arg Glu Ala Ser Pro Arg Leu Arg Val His Lys                   80 85 90  Thr Phe Lys Phe Val Val Val Gly Val Leu Leu Gln Val Val Pro                   95 100 105  Ser Ser Ala Ala Thr Ile Lys Leu His Asp Gln Ser Ile Gly Thr                  110 115 120  Gln Gln Trp Glu His Ser Pro Leu Gly Glu Leu Cys Pro Pro Gly                  125 130 135  Ser His Arg Ser Glu Arg Pro Gly Ala Cys Asn Arg Cys Thr Glu                  140 145 150  Gly Val Gly Tyr Thr Asn Ala Ser Asn Asn Leu Phe Ala Cys Leu                  155 160 165  Pro Cys Thr Ala Cys Lys Ser Asp Glu Glu Glu Arg Ser Pro Cys                  170 175 180  Thr Thr Thr Arg Asn Thr Ala Cys Gln Cys Lys Pro Gly Thr Phe                  185 190 195  Arg Asn Asp Asn Ser Ala Glu Met Cys Arg Lys Cys Ser Thr Gly                  200 205 210  Cys Pro Arg Gly Met Val Lys Val Lys Asp Cys Thr Pro Trp Ser                  215 220 225  Asp Ile Glu Cys Val His Lys Glu Ser Gly Asn Gly His Asn Ile                  230 235 240  Trp Val Ile Leu Val Val Thr Leu Val Val Pro Leu Leu Leu Val                  245 250 255  Ala Val Leu Ile Val Cys Cys Cys Ile Gly Ser Gly Cys Gly Gly                  260 265 270  Asp Pro Lys Cys Met Asp Arg Val Cys Phe Trp Arg Leu Gly Leu                  275 280 285  Leu Arg Gly Pro Gly Ala Glu Asp Asn Ala His Asn Glu Ile Leu                  290 295 300  Ser Asn Ala Asp Ser Leu Ser Thr Phe Val Ser Glu Gln Gln Met                  305 310 315  Glu Ser Gln Glu Pro Ala Asp Leu Thr Gly Val Thr Val Gln Ser                  320 325 330  Pro Gly Glu Ala Gln Cys Leu Leu Gly Pro Ala Glu Ala Glu Gly                  335 340 345  Ser Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Gly Ala Asp Pro                  350 355 360  Thr Glu Thr Leu Met Leu Phe Phe Asp Lys Phe Ala Asn Ile Val                  365 370 375  Pro Phe Asp Ser Trp Asp Gln Leu Met Arg Gln Leu Asp Leu Thr                  380 385 390  Lys Asn Glu Ile Asp Val Val Arg Ala Gly Thr Ala Gly Pro Gly                  395 400 405  Asp Ala Leu Tyr Ala Met Leu Met Lys Trp Val Asn Lys Thr Gly                  410 415 420  Arg Asn Ala Ser Ile His Thr Leu Leu Asp Ala Leu Glu Arg Met                  425 430 435  Glu Glu Arg His Ala Lys Glu Lys Ile Gln Asp Leu Leu Val Asp                  440 445 450  Ser Gly Lys Phe Ile Tyr Leu Glu Asp Gly Thr Gly Ser Ala Val                  455 460 465  Ser leu glu              <210> 4 <211> 1407 <212> DNA <213> Homo sapiens <400> 4  atggcgccac caccagctag agtacatcta ggtgcgttcc tggcagtgac 50  tccgaatccc gggagcgcag cgagtgggac agaggcagcc gcggccacac 100  ccagcaaagt gtggggctct tccgcgggga ggattgaacc acgaggcggg 150  ggccgaggag cgctccctac ctccatggga cagcacggac ccagtgcccg 200  ggcccgggca gggcgcgccc caggacccag gccggcgcgg gaagccagcc 250  ctcggctccg ggtccacaag accttcaagt ttgtcgtcgt cggggtcctg 300  ctgcaggtcg tacctagctc agctgcaacc atgatcaatc aattggcaca 350  aattggcaca cagcaatggg aacatagccc tttgggagag ttgtgtccac 400  caggatctca tagatcagaa cgtcctggag cctgtaaccg gtgcacagag 450  ggtgtgggtt acaccaatgc ttccaacaat ttgtttgctt gcctcccatg 500  tacagcttgt aaatcagatg aagaagagag aagtccctgc accacgacca 550  ggaacacagc atgtcagtgc aaaccaggaa ctttccggaa tgacaattct 600  gctgagatgt gccggaagtg cagcacaggg tgccccagag ggatggtcaa 650  ggtcaaggat tgtacgccct ggagtgacat cgagtgtgtc cacaaagaat 700  caggcaatgg acataatata tgggtgattt tggttgtgac tttggttgtt 750  ccgttgctgt tggtggctgt gctgattgtc tgttgttgca tcggctcagg 800  ttgtggaggg gaccccaagt gcatggacag ggtgtgtttc tggcgcttgg 850  gtctcctacg agggcctggg gctgaggaca atgctcacaa cgagattctg 900  agcaacgcag actcgctgtc cactttcgtc tctgagcagc aaatggaaag 950  ccaggagccg gcagatttga caggtgtcac tgtacagtcc ccaggggagg 1000  cacagtgtct gctgggaccg gcagaagctg aagggtctca gaggaggagg 1050  ctgctggttc cagcaaatgg tgctgacccc actgagactc tgatgctgtt 1100  ctttgacaag tttgcaaaca tcgtgccctt tgactcctgg gaccagctca 1150  tgaggcagct ggacctcacg aaaaatgaga tcgatgtggt cagagctggt 1200  acagcaggcc caggggatgc cttgtatgca atgctgatga aatgggtcaa 1250  caaaactgga cggaacgcct cgatccacac cctgctggat gccttggaga 1300  ggatggaaga gagacatgca aaagagaaga ttcaggacct cttggtggac 1350  tctggaaagt tcatctactt agaagatggc acaggctctg ccgtgtcctt 1400  ggagtga 1407 <210> 5 <211> 411 <212> PRT <213> Homo sapiens <400> 5  Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg    1 5 10 15  Lys Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro                   20 25 30  Gly Leu Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val                   35 40 45  Leu Leu Leu Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp                   50 55 60  Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser                   65 70 75  Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile Ser Glu Asp                   80 85 90  Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr                   95 100 105  His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp                  110 115 120  Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr                  125 130 135  Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro                  140 145 150  Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val                  155 160 165  Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His                  170 175 180  Lys Glu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val                  185 190 195  Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys                  200 205 210  Val Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp                  215 220 225  Pro Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp                  230 235 240  Asn Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val                  245 250 255  Pro Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly                  260 265 270  Val Asn Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu Pro                  275 280 285  Ala Glu Ala Glu Arg Ser Gln Arg Arg Arg Leu Leu Val Pro Ala                  290 295 300  Asn Glu Gly Asp Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp                  305 310 315  Phe Ala Asp Leu Val Pro Phe Asp Ser Trp Glu Pro Leu Met Arg                  320 325 330  Lys Leu Gly Leu Met Asp Asn Glu Ile Lys Val Ala Lys Ala Glu                  335 340 345  Ala Ala Gly His Arg Asp Thr Leu Tyr Thr Met Leu Ile Lys Trp                  350 355 360  Val Asn Lys Thr Gly Arg Asp Ala Ser Val His Thr Leu Leu Asp                  365 370 375  Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln Lys Ile Glu                  380 385 390  Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu Gly Asn                  395 400 405  Ala Asp Ser Ala Leu Ser                  410 <210> 6 <211> 440 <212> PRT <213> Homo sapiens <400> 6  Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg    1 5 10 15  Lys Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro                   20 25 30  Gly Pro Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val                   35 40 45  Leu Leu Leu Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp                   50 55 60  Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser                   65 70 75  Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile Ser Glu Asp                   80 85 90  Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr                   95 100 105  His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp                  110 115 120  Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr                  125 130 135  Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro                  140 145 150  Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val                  155 160 165  Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His                  170 175 180  Lys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro Ala Val Glu                  185 190 195  Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro Cys Ser                  200 205 210  Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val Leu                  215 220 225  Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val                  230 235 240  Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro                  245 250 255  Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn                  260 265 270  Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro                  275 280 285  Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val                  290 295 300  Asn Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala                  305 310 315  Glu Ala Glu Arg Ser Gln Arg Arg Arg Leu Leu Val Pro Ala Asn                  320 325 330  Glu Gly Asp Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe                  335 340 345  Ala Asp Leu Val Pro Phe Asp Ser Trp Glu Pro Leu Met Arg Lys                  350 355 360  Leu Gly Leu Met Asp Asn Glu Ile Lys Val Ala Lys Ala Glu Ala                  365 370 375  Ala Gly His Arg Asp Thr Leu Tyr Thr Met Leu Ile Lys Trp Val                  380 385 390  Asn Lys Thr Gly Arg Asp Ala Ser Val His Thr Leu Leu Asp Ala                  395 400 405  Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln Lys Ile Glu Asp                  410 415 420  His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu Gly Asn Ala                  425 430 435  Asp Ser Ala Met Ser                  440 <210> 7 <211> 1180 <212> DNA <213> Homo sapiens <400> 7  gctgtgggaa cctctccacg cgcacgaact cagccaacga tttctgatag 50  atttttggga gtttgaccag agatgcaagg ggtgaaggag cgcttcctac 100  cgttagggaa ctctggggac agagcgcccc ggccgcctga tggccgaggc 150  agggtgcgac ccaggaccca ggacggcgtc gggaaccata ccatggcccg 200  gatccccaag accctaaagt tcgtcgtcgt catcgtcgcg gtcctgctgc 250  cagtcctagc ttactctgcc accactgccc ggcaggagga agttccccag 300  cagacagtgg ccccacagca acagaggcac agcttcaagg gggaggagtg 350  tccagcagga tctcatagat cagaacatac tggagcctgt aacccgtgca 400  cagagggtgt ggattacacc aacgcttcca acaatgaacc ttcttgcttc 450  ccatgtacag tttgtaaatc agatcaaaaa cataaaagtt cctgcaccat 500  gaccagagac acagtgtgtc agtgtaaaga aggcaccttc cggaatgaaa 550  actccccaga gatgtgccgg aagtgtagca ggtgccctag tggggaagtc 600  caagtcagta attgtacgtc ctgggatgat atccagtgtg ttgaagaatt 650  tggtgccaat gccactgtgg aaaccccagc tgctgaagag acaatgaaca 700  ccagcccggg gactcctgcc ccagctgctg aagagacaat gaacaccagc 750  ccagggactc ctgccccagc tgctgaagag acaatgacca ccagcccggg 800  gactcctgcc ccagctgctg aagagacaat gaccaccagc ccggggactc 850  ctgccccagc tgctgaagag acaatgacca ccagcccggg gactcctgcc 900  tcttctcatt acctctcatg caccatcgta gggatcatag ttctaattgt 950  gcttctgatt gtgtttgttt gaaagacttc actgtggaag aaattccttc 1000  cttacctgaa aggttcaggt aggcgctggc tgagggcggg gggcgctgga 1050  cactctctgc cctgcctccc tctgctgtgt tcccacagac agaaacgcct 1100  gcccctgccc caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1150  aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1180 <210> 8 <211> 259 <212> PRT <213> Homo sapiens <400> 8  Met Ala Arg Ile Pro Lys Thr Leu Lys Phe Val Val Val Ile Val    1 5 10 15  Ala Val Leu Leu Pro Val Leu Ala Tyr Ser Ala Thr Thr Ala Arg                   20 25 30  Gln Glu Glu Val Pro Gln Gln Thr Val Ala Pro Gln Gln Gln Arg                   35 40 45  His Ser Phe Lys Gly Glu Glu Cys Pro Ala Gly Ser His Arg Ser                   50 55 60  Glu His Thr Gly Ala Cys Asn Pro Cys Thr Glu Gly Val Asp Tyr                   65 70 75  Thr Asn Ala Ser Asn Asn Glu Pro Ser Cys Phe Pro Cys Thr Val                   80 85 90  Cys Lys Ser Asp Gln Lys His Lys Ser Ser Cys Thr Met Thr Arg                   95 100 105  Asp Thr Val Cys Gln Cys Lys Glu Gly Thr Phe Arg Asn Glu Asn                  110 115 120  Ser Pro Glu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly Glu                  125 130 135  Val Gln Val Ser Asn Cys Thr Ser Trp Asp Asp Ile Gln Cys Val                  140 145 150  Glu Glu Phe Gly Ala Asn Ala Thr Val Glu Thr Pro Ala Ala Glu                  155 160 165  Glu Thr Met Asn Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu                  170 175 180  Glu Thr Met Asn Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu                  185 190 195  Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu                  200 205 210  Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu                  215 220 225  Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Ser Ser His Tyr                  230 235 240  Leu Ser Cys Thr Ile Val Gly Ile Ile Val Leu Ile Val Leu Leu                  245 250 255  Ile Val Phe Val                  <210> 9 <211> 2082 <212> DNA <213> Homo sapiens <400> 9  ccaactgcac ctcggttcta tcgattgaat tccccgggga tcctctagag 50  atccctcgac ctcgacccac gcgtccggaa cctttgcacg cgcacaaact 100  acggggacga tttctgattg atttttggcg ctttcgatcc accctcctcc 150  cttctcatgg gactttgggg acaaagcgtc ccgaccgcct cgagcgctcg 200  agcagggcgc tatccaggag ccaggacagc gtcgggaacc agaccatggc 250  tcctggaccc caagatcctt aagttcgtcg tcttcatcgt cgcggttctg 300  ctgccggtcc gggttgactc tgccaccatc ccccggcagg acgaagttcc 350  ccagcagaca gtggccccac agcaacagag gcgcagcctc aaggaggagg 400  agtgtccagc aggatctcat agatcagaat atactggagc ctgtaacccg 450  tgcacagagg gtgtggatta caccattgct tccaacaatt tgccttcttg 500  cctgctatgt acagtttgta aatcaggtca aacaaataaa agttcctgta 550  ccacgaccag agacaccgtg tgtcagtgtg aaaaaggaag cttccaggat 600  aaaaactccc ctgagatgtg ccggacgtgt agaacagggt gtcccagagg 650  gatggtcaag gtcagtaatt gtacgccccg gagtgacatc aagtgcaaaa 700  atgaatcagc tgccagttcc actgggaaaa ccccagcagc ggaggagaca 750  gtgaccacca tcctggggat gcttgcctct ccctatcact accttatcat 800  catagtggtt ttagtcatca ttttagctgt ggttgtggtt ggcttttcat 850  gtcggaagaa attcatttct tacctcaaag gcatctgctc aggtggtgga 900  ggaggtcccg aacgtgtgca cagagtcctt ttccggcggc gttcatgtcc 950  ttcacgagtt cctggggcgg aggacaatgc ccgcaacgag accctgagta 1000  acagatactt gcagcccacc caggtctctg agcaggaaat ccaaggtcag 1050  gagctggcag agctaacagg tgtgactgta gagtygccag aggagccaca 1100  gcgtctgctg gaacaggcag aagctgaagg gtgtcagagg aggaggctgc 1150  tggttccagt gaatgacgct gactccgctg acatcagcac cttgctggat 1200  gcctcggcaa cactggaaga aggacatgca aaggaaacaa ttcaggacca 1250  actggtgggc tccgaaaagc tcttttatga agaagatgag gcaggctctg 1300  ctacgtcctg cctgtgaaag aatctcttca ggaaaccaga gcttccctca 1350  tttacctttt ctcctacaaa gggaagcagc ctggaagaaa cagtccagta 1400  cttgacccat gccccaacaa actctactat ccaatatggg gcagcttacc 1450  aatggtccta gaactttgtt aacgcacttg gagtaatttt tatgaaatac 1500  tgcgtgtgat aagcaaacgg gagaaattta tatcagattc ttggctgcat 1550  agttatacga ttgtgtatta agggtcgttt taggccacat gcggtggctc 1600  atgcctgtaa tcccagcact ttgataggct gaggcaggtg gattgcttga 1650  gctcgggagt ttgagaccag cctcatcaac acagtgaaac tccatctcaa 1700  tttaaaaaga aaaaaagtgg ttttaggatg tcattctttg cagttcttca 1750  tcatgagaca agtctttttt tctgcttctt atattgcaag ctccatctct 1800  actggtgtgt gcatttaatg acatctaact acagatgccg cacagccaca 1850  atgctttgcc ttatagtttt ttaactttag aacgggatta tcttgttatt 1900  acctgtattt tcagtttcgg atatttttga cttaatgatg agattatcaa 1950  gacgtacccc tatgctaagt catgagcata tggacttacg agggttcgac 2000  ttagagtttt gagctttaag ataggattat tgggggctta cccccacctt 2050  aattagaaga aacattttat attgctttac ta 2082 <210> 10 <211> 386 <212> PRT <213> Homo sapiens <220> <221> Unsure <222> 310 <223> Unknown amino acid <400> 10  Met Gly Leu Trp Gly Gln Ser Val Pro Thr Ala Ser Ser Ala Arg    1 5 10 15  Ala Gly Arg Tyr Pro Gly Ala Arg Thr Ala Ser Gly Thr Arg Pro                   20 25 30  Trp Leu Leu Asp Pro Lys Ile Leu Lys Phe Val Val Phe Ile Val                   35 40 45  Ala Val Leu Leu Pro Val Arg Val Asp Ser Ala Thr Ile Pro Arg                   50 55 60  Gln Asp Glu Val Pro Gln Gln Thr Val Ala Pro Gln Gln Gln Arg                   65 70 75  Arg Ser Leu Lys Glu Glu Glu Cys Pro Ala Gly Ser His Arg Ser                   80 85 90  Glu Tyr Thr Gly Ala Cys Asn Pro Cys Thr Glu Gly Val Asp Tyr                   95 100 105  Thr Ile Ala Ser Asn Asn Leu Pro Ser Cys Leu Leu Cys Thr Val                  110 115 120  Cys Lys Ser Gly Gln Thr Asn Lys Ser Ser Cys Thr Thr Thr Arg                  125 130 135  Asp Thr Val Cys Gln Cys Glu Lys Gly Ser Phe Gln Asp Lys Asn                  140 145 150  Ser Pro Glu Met Cys Arg Thr Cys Arg Thr Gly Cys Pro Arg Gly                  155 160 165  Met Val Lys Val Ser Asn Cys Thr Pro Arg Ser Asp Ile Lys Cys                  170 175 180  Lys Asn Glu Ser Ala Ala Ser Ser Thr Gly Lys Thr Pro Ala Ala                  185 190 195  Glu Glu Thr Val Thr Thr Ile Leu Gly Met Leu Ala Ser Pro Tyr                  200 205 210  His Tyr Leu Ile Ile Ile Val Val Leu Val Ile Ile Leu Ala Val                  215 220 225  Val Val Val Gly Phe Ser Cys Arg Lys Lys Phe Ile Ser Tyr Leu                  230 235 240  Lys Gly Ile Cys Ser Gly Gly Gly Gly Gly Pro Glu Arg Val His                  245 250 255  Arg Val Leu Phe Arg Arg Arg Ser Cys Pro Ser Arg Val Pro Gly                  260 265 270  Ala Glu Asp Asn Ala Arg Asn Glu Thr Leu Ser Asn Arg Tyr Leu                  275 280 285  Gln Pro Thr Gln Val Ser Glu Gln Glu Ile Gln Gly Gln Glu Leu                  290 295 300  Ala Glu Leu Thr Gly Val Thr Val Glu Xaa Pro Glu Glu Pro Gln                  305 310 315  Arg Leu Leu Glu Gln Ala Glu Ala Glu Gly Cys Gln Arg Arg Arg                  320 325 330  Leu Leu Val Pro Val Asn Asp Ala Asp Ser Ala Asp Ile Ser Thr                  335 340 345  Leu Leu Asp Ala Ser Ala Thr Leu Glu Glu Gly His Ala Lys Glu                  350 355 360  Thr Ile Gln Asp Gln Leu Val Gly Ser Glu Lys Leu Phe Tyr Glu                  365 370 375  Glu Asp Glu Ala Gly Ser Ala Thr Ser Cys Leu                  380 385 <210> 11 <211> 2742 <212> DNA <213> Homo sapiens <400> 11  cccaacccac gatggtctgg gagctgcgcc cagggcttgg cgctggcggc 50  cccgcaacag caccgagcgt ttcggtcggc gtcgggcccg ccctccccgc 100  tcactccctc cgccctcgtg ctcctcccgg ggtgcttggc acagcctcgg 150  attcctccct gtggcggcgt ggggaacatc tcggcagcca ccgcgcttct 200  cccgctggag cgggcgtcca gcttggctgc cctcggtcct cttgcaggat 250  cctgccgccc ctccaaccgg atcctgggtc tagagctccc cagagcgagg 300  cgctcgccag gactcctgcc atgcggcgcc tgactcgtcg gctggttctg 350  ccagtcttcg gggtgctctg gatcacggtg ctgctgttct tctgggtaac 400  gacctgtggg accagtttga tgagcggcgg tatctgaatg ccaaaaagtg 450  gcgcgttggt gacgacccct ataagctgta tgcacactgc tggtgtattg 500  cacggacctt ccacccacta gcatcatcat caccttccac aacgaggccc 550  gctccacgct gacttcagca atgatcctga tgactgtaaa cagctcatca 600  agttgcccaa ggtgaaatgc ttgcgcaata atgaacggca acactgtgag 650  gtgaacaggg actggctcca gcctctgttg cacagggtca aagaggacta 700  cacgcgggtg gtgtgccctg ttggagcctc cacttccagt gggagcagct 750  ctccccagag cagaaggctc ggcgcctgga ccccacggag cccatcagga 800  cggcatcccg agtgcaccgc tcccgccccg ccccgccttg cgggcggcgg 850  tagcgccccc tctcagagcc ccgctcactc ccacctcggc tcgctccgag 900  tcggcctgtc tctcgctgct cgagtcagtt tccctatcgg cggcagcggg 950  caaggcggcg gcggcggcgg cggcagccgc gtccctgcca cgtttcgggt 1000  cgccctgcac cccccaccca ggctcgcttc tcttcgaagc gggaagggcg 1050  cccgccaacc ctgaccgccg gggggtgccc ccgggacgta gcgccgcgga 1100  gaggaagcgg caaaggggac ccaagaggaa gttggaggtg ccgacgggac 1150  ctgaagtgca gacccctaag ccttcggacg ctgactggga ctgctttcaa 1200  ccagcgggag agtgagcgga tctccagcaa tcgggccatc ccggacactc 1250  gccatctgag agctcaggac catccgcagt gtattaaacc gcacccctac 1300  gcatctgatc cgggaaatca tattagtgga taggtctggt ccggtcccgg 1350  attcggggcg ctgacatcgc ccagggcacc actctgactt tcctcgacag 1400  cgatcgatat cattaacctg gacaccttca cctacatcga gtctgcctcg 1450  gagctcagag gggggtttga ctcctatcat agctggaggg ctcttcgtga 1500  tcgacaaagc ttggtttgat tacctgggga aatatgatat ggacatggac 1550  atctggggtg gggagaactt tgaaatctcc ttccgagtgt ggatgtgcgg 1600  gggcagccta gagatcgtcc ctatataaag aacaccaagc ggacagctga 1650  agtgtggatg gatgaataca agcaatacta ttacgctgcc cggccattcg 1700  caagtggtac ctggagaata tctaccctga actcagcatc cccaaggagt 1750  cctccatcca gaagggcaat atccgacaga gaaggtcaaa ggcgaagatg 1800  caaagtccca ggtatgggcc ttcacataca cccagcagat cctccaggag 1850  gagctgtgcc tcaatggacc aaaactggtt cccacatcga gcacatagca 1900  tcccacctct gcctcgatac agatatgttc ggtgatggca cagctcttga 1950  ggacccctgc cagaagcagc aagggccatg gggtggtgct tccctggacc 2000  agaacagact ggaaactggg ccaactgtct cagggaggac agaggaaaac 2050  atcacaagcc aatggggctc aaagacaaat cccacatgtt ctcaaggccg 2100  ttgttccttt tcctacaaag gaagcagtct ctggaggcca gaaacaaaag 2150  ccttcttttt cactaggcca ggactacatt gactgcagcc gagtggggca 2200  cgtcttccgg aagaagcacc cctacgtttt ccctgatgga aatgccaaca 2250  cgcctggaga ggcccttcgg gaatgttgag agcagattgg acctgaggaa 2300  gaatctgcgc tgccagagct tcacagaagt gcctggaatc tcaaaggcag 2350  aacaaccaag aaaccccaaa cctaaagttg agcccctgtg ccgtcagtca 2400  tcaccttgtt ccctggcgcc ccagtggttc ttgtcctttg caagaatgga 2450  gatgaccgac agcgagaacg gcaaggaaat cgtcgtcaac ccatgtgagt 2500  cctcactcat gagccagcac tgggacatgg tgagcaagca gcctgcaacc 2550  acctcagaca tcctggactg ggaggtccag gcagagcccc ccaggacagg 2600  agtaagttcc agtcctggcc agtcattccc tgattggtat ctggagacag 2650  aaacctaatg ggaagtgttt atagagatga agaatggagg ttgtttccaa 2700  aagaaataaa gagaaactta gaagttgaaa aaaaaaaaaa aa 2742 <210> 12 <211> 552 <212> PRT <213> Homo sapiens <400> 12  Met Arg Arg Leu Thr Arg Arg Leu Val Leu Pro Val Phe Gly Val    1 5 10 15  Leu Trp Ile Thr Val Leu Leu Phe Phe Trp Val Thr Asp Leu Trp                   20 25 30  Asp Gln Phe Asp Glu Arg Arg Tyr Leu Asn Ala Lys Lys Trp Arg                   35 40 45  Val Gly Asp Asp Pro Tyr Lys Leu Tyr Cys Thr Leu Leu Val Tyr                   50 55 60  Cys Thr Asp Leu Pro Pro Thr Ser Ile Ile Thr Phe His Asn                   65 70 75  Glu Ala Arg Ser Thr Leu Asp Phe Ser Asn Asp Pro Asp Asp Cys                   80 85 90  Lys Gln Leu Ile Lys Leu Pro Lys Val Lys Cys Leu Arg Asn Asn                   95 100 105  Glu Arg Gln His Cys Glu Val Asn Arg Asp Trp Leu Gln Pro Leu                  110 115 120  Leu His Arg Val Lys Glu Asp Tyr Thr Arg Val Val Cys Pro Val                  125 130 135  Trp Ser Leu His Phe Gln Trp Glu Gln Leu Ser Pro Glu Gln Lys                  140 145 150  Ala Arg Arg Leu Asp Pro Thr Glu Pro Ile Arg Thr Lys Arg Lys                  155 160 165  Leu Glu Val Pro Thr Gly Pro Glu Val Gln Thr Pro Lys Pro Ser                  170 175 180  Asp Ala Asp Trp Asp Ala Phe Asn Gln Arg Glu Ser Glu Arg Ile                  185 190 195  Ser Ser Asn Arg Ala Ile Pro Asp Thr Arg His Leu Arg Leu Arg                  200 205 210  Thr Ile Arg Ser Val Leu Asn Arg Thr Pro Thr His Leu Ile Arg                  215 220 225  Glu Ile Ile Leu Val Asp Gly Leu Val Arg Ser Arg Ile Arg Gly                  230 235 240  Ala Asp Ile Ala Gln Gly Thr Thr Leu Thr Phe Leu Asp Ser Ile                  245 250 255  Asp Ile Ile Asn Leu Asp Thr Phe Thr Tyr Ile Glu Ser Ala Ser                  260 265 270  Glu Leu Arg Gly Gly Phe Asp Pro Ile Ile Ala Gly Gly Leu Phe                  275 280 285  Val Ile Asp Lys Ala Trp Phe Asp Tyr Leu Gly Lys Tyr Asp Met                  290 295 300  Asp Met Asp Ile Trp Gly Gly Glu Asn Phe Glu Ile Ser Phe Arg                  305 310 315  Val Trp Met Cys Gly Gly Ser Leu Glu Ile Val Pro Tyr Ile Lys                  320 325 330  Asn Thr Lys Arg Thr Ala Glu Val Trp Met Asp Glu Tyr Lys Gln                  335 340 345  Tyr Tyr Tyr Ala Ala Arg Pro Phe Ala Lys Trp Tyr Leu Glu Asn                  350 355 360  Ile Tyr Pro Glu Leu Ser Ile Pro Lys Glu Ser Ser Ile Gln Lys                  365 370 375  Gly Asn Ile Arg Gln Arg Lys Val Lys Gly Glu Asp Ala Lys Ser                  380 385 390  Gln Val Trp Ala Phe Thr Tyr Thr Gln Gln Ile Leu Gln Glu Glu                  395 400 405  Leu Cys Leu Gln Trp Thr Lys Thr Gly Ser His Ile Glu His Ile                  410 415 420  Ala Ser His Leu Cys Leu Asp Thr Asp Met Phe Gly Asp Gly Thr                  425 430 435  Ser Ser Cys Ser Arg Val Gly His Val Phe Arg Lys Lys His Pro                  440 445 450  Tyr Val Phe Pro Asp Gly Asn Ala Asn Thr Leu Glu Arg Pro Phe                  455 460 465  Gly Asn Val Glu Ser Arg Leu Asp Leu Arg Lys Asn Leu Arg Cys                  470 475 480  Gln Ser Phe Gln Lys Cys Leu Glu Ser Gln Arg Gln Asn Asn Gln                  485 490 495  Glu Thr Pro Asn Leu Lys Leu Ser Pro Cys Ala Ser Val Ile Thr                  500 505 510  Leu Phe Pro Gly Ala Pro Val Val Leu Val Leu Cys Lys Asn Gly                  515 520 525  Asp Asp Arg Gln Glu Asn Gly Lys Glu Ile Val Val Asn Pro Cys                  530 535 540  Glu Ser Ser Leu Met Ser Gln His Trp Asp Met Val                  545 550 <210> 13 <211> 2937 <212> DNA <213> Homo sapiens <400> 13  atggctcacc taaagcgact agtaaaatta cacattaaaa gacattacca 50  taaaaagttc tggaagcttg gtgcagtaat aggatggaaa ggaacatgaa 100  aaacaaaaac aagatgttgg atttaatgct agaagctgta aacaatatta 150  aggatgccat tattatacag cagcagaatt gaagcctgtc cttgaccgtc 200  cacctcagga ttcaaatgca cctggtgctt ctggtaaagc aatgctttcg 250  caagtgacag gatttctttg caccgagatc ttggaccaga cactcgacct 300  cctgaatgta ttgaacaaaa ttgcttagaa ctgtccacag tgtgctctat 350  tcttcacctg caatactgct gaaggaaatc attttggtgg atgatgctag 400  caaagagaaa gaaaaggtct gatcactgct cggttgctag gagcaacagt 450  cgcaacagct gaaacgctca catttttaga gtcgtaagtc cagatattgc 500  atccatagat ctgaacacgt ttgaattcaa caaaccttct ccttatggaa 550  gtaaccataa aaagatgaaa cctacccaat taaaacaccc acttttgcag 600  gaggactttt ttccatatca aaagaatatt ttgagtatat tgtggtgggc 650  agttggagat tatgccttgc tctgttgttg gacatgtttt tcgcagcaaa 700  agccctcata gctttccaaa ttttatagga gaaatacaga tgcagcaaaa 750  attgttaaac aaaaagcatt tggtgatctt tcaaaaagat ttgaaataaa 800  gaagaagaaa taactgttat ttgtcaagtg acaagctttt aatgtcagat 850  tttttcttta taatagtttt ggttttaatg caaagagaag taagtgttca 900  atattccaaa gaggaatcag ccaaaaatgc aaataggagc acctgtcagg 950  caaaacattg atgctggtga gagaccttgt ttgcaaggaa ttcaagacaa 1000  ccaatttaag tgttgaagag caaaaggaaa aggaacgtgg ggaagctaaa 1050  cactgcttta tttaagcgct gccctcccct gcccaccacc agtgtcataa 1100  tagtttttca taatgaagcg tggtccacgt gtagatgagt acttacatga 1150  taaactagat gaatatgtaa aacaattttc tatagtaaaa atagtcagat 1200  gctcactgtg agtgtttcta tggttggcta gaacctctgt tggccagaat 1250  agctgagaac tacacggctc cgtggaaatt ttgactggag tctttcattt 1300  ggctgggagt cgcttcctga tcatgagaag caaagaaggt ggaagctatg 1350  atgaagaaat ggaaatctgg ggaggtgaaa atatagaaat gtctttcaga 1400  gtatggcaaa ggcactcagg tgattgctag aaaccaagtt cgccttgcag 1450  aagtctggat ggatgaatac aaggaaataa caccgtcttc ggtgtaaaaa 1500  ttttacatgg tatctgaaca acatttatcc agaggtgtat gtgccagacc 1550  ttaatcctgt tatatctgga tacattaaaa gcgttggtca gcctctatgt 1600  ctggatgttg gagaaaacaa tcaaggaggg aaattcggca caacatccag 1650  aaggaattat gtcttcatgc tgctcaaggt ctcgttcagc tgaaggcatg 1700  tacctacaat tcttaaaaat gtgcctttca gcaaatggag agcatccaag 1750  tttagtgtca tgcaacccat cagatccact ccaaaaatgc tgtgactagg 1800  catacactgt agtttttgaa aattatgcaa aagcagctaa atgtaactta 1850  ttccaagtgc atttttctta taccaaagac tatttcaaaa tgtccagatg 1900  taggggaaga gatgtttaca gtatgatgaa aataattttc caagtaaagt 1950  atttccccta gttttttggg gggataggaa gaaagatttg ttactgtatt 2000  tttttaacta cataaaaata gatcaataag gtgaactttt ttttgcgttt 2050  ggtttacttg tctgtcaaat gtttccttaa acatgaaact gaataaggag 2100  aagagtattt aagtcttcct taaatgactt ttcttaagta atgatactgt 2150  gtgttttccc aaagcacttt taaaaaaatt tttataaatt gaatgtttgt 2200  gatattaaat ttcaaatgca gaatacttga ctcatttaaa gctaaatttt 2250  gttactgatt caattataaa acacaataaa aaatcctcaa cactaaaaaa 2300  aaaaaaacaa accattaatt atgtatacat gtcatggact tgggggaaac 2350  cagtactttg aatactctgc tcaacatagg tcacaagaca gttgtcactg 2400  gagagcagat atgggagatc cagaaggatc aacttctata caatccagat 2450  acttagccaa aatgattaag tgttccttaa aattaagttg aaaaaggaaa 2500  tattctttct cataaaaatt tatatcttta tgtagcacta tctacagaaa 2550  ttctgcaagt ttctgtttca aagcacaata actagtatga agtttgtgtg 2600  ttttgtacac ttagggatat atatatatag ctacattcac acactcacaa 2650  tttaaaaatg tcagcattgg cctctgtgta caaaccaaga gcttttacag 2700  atccagaatt tattagttta aaatgcattt aacacttaaa tttcttggca 2750  aattttaaaa cattttttag tctgtaatac actccacttg aagcacttac 2800  tatctgttga aaaggtgtcc ttttcctttc ttctagtatt ttttttctta 2850  ccaaaattca ctaatctttg taatggattt ttgactttgt aatggattct 2900  tttcatcaaa aagccttatt tttttatcta tgtggaa 2937 <210> 14 <211> 633 <212> PRT <213> Homo sapiens <400> 14  Met Ala His Leu Lys Arg Leu Val Lys Leu His Ile Lys Arg His    1 5 10 15  Tyr His Lys Lys Phe Trp Lys Leu Gly Ala Val Ile Arg Met Glu                   20 25 30  Arg Asn Met Lys Asn Lys Asn Lys Met Leu Asp Leu Met Leu Glu                   35 40 45  Ala Val Asn Asn Ile Lys Asp Ala Met Tyr Tyr Thr Ala Ala Glu                   50 55 60  Leu Lys Pro Val Leu Asp Arg Pro Pro Gln Asp Ser Asn Ala Pro                   65 70 75  Gly Ala Ser Gly Lys Ala Asn Ala Phe Ala Ser Asp Arg Ile Ser                   80 85 90  Leu His Arg Asp Leu Gly Pro Asp Thr Arg Pro Pro Glu Cys Ile                   95 100 105  Glu Gln Lys Leu Leu Arg Thr Val His Ser Val Leu Tyr Ser Ser                  110 115 120  Pro Ala Ile Leu Leu Lys Glu Ile Ile Leu Val Asp Asp Ala Ser                  125 130 135  Gln Arg Glu Arg Lys Gly Leu Ile Thr Ala Arg Leu Leu Gly Ala                  140 145 150  Thr Val Ala Thr Ala Glu Thr Leu Thr Phe Leu Asp Val Val Ser                  155 160 165  Pro Asp Ile Ala Ser Ile Asp Leu Asn Thr Phe Glu Phe Asn Lys                  170 175 180  Pro Ser Pro Tyr Gly Ser Asn His Asn Lys Asp Glu Thr Tyr Pro                  185 190 195  Ile Lys Thr Pro Thr Phe Ala Gly Gly Leu Phe Ser Ile Ser Lys                  200 205 210  Glu Tyr Phe Glu Tyr Ile Cys Gly Gly Gln Leu Glu Ile Met Pro                  215 220 225  Cys Ser Val Val Gly Asn Val Phe Arg Ser Lys Ser Pro Asn Ser                  230 235 240  Phe Pro Lys Phe Tyr Arg Arg Asn Thr Asp Ala Ala Lys Ile Val                  245 250 255  Lys Gln Lys Ala Phe Gly Asp Leu Ser Lys Arg Phe Glu Ile Lys                  260 265 270  Phe Phe Phe Ile Ile Val Leu Val Leu Met Gln Arg Glu Val Ser                  275 280 285  Val Gln Tyr Ser Lys Glu Glu Ser Pro Lys Met Gln Ile Gly Ala                  290 295 300  Pro Val Arg Gln Asn Ile Asp Ala Gly Glu Arg Pro Cys Leu Gln                  305 310 315  Gly Phe Lys Thr Thr Asn Leu Ser Val Glu Glu Gln Lys Glu Lys                  320 325 330  Glu Arg Gly Glu Ala Lys His Cys Phe Phe Lys Arg Cys Pro Pro                  335 340 345  Leu Pro Thr Thr Ser Val Ile Ile Val Phe His Asn Glu Ala Trp                  350 355 360  Ser Thr Val Asp Glu Tyr Leu His Asp Lys Leu Asp Glu Tyr Val                  365 370 375  Lys Gln Phe Ser Ile Val Lys Ile Val Arg Ala His Cys Glu Cys                  380 385 390  Phe Tyr Gly Trp Leu Glu Pro Leu Leu Ala Arg Ile Ala Glu Asn                  395 400 405  Tyr Thr Ala Arg Gly Asn Phe Asp Trp Ser Leu Ser Phe Gly Trp                  410 415 420  Glu Ser Leu Pro Asp His Glu Lys Gln Arg Arg Gly Ser Tyr Asp                  425 430 435  Glu Glu Met Glu Ile Trp Gly Gly Glu Asn Ile Glu Met Ser Pro                  440 445 450  Arg Val Trp Gln Gly Thr Gln Val Ile Ala Arg Asn Gln Val Arg                  455 460 465  Leu Ala Glu Val Trp Met Asp Glu Tyr Lys Glu Ile His Arg Leu                  470 475 480  Arg Cys Lys Asn Phe Thr Trp Tyr Leu Asn Asn Ile Tyr Pro Glu                  485 490 495  Val Tyr Val Pro Asp Leu Asn Pro Val Ile Ser Gly Tyr Ile Lys                  500 505 510  Ser Val Gly Gln Pro Leu Cys Leu Asp Val Gly Glu Asn Asn Gln                  515 520 525  Gly Gly Glu Ile Arg His Asn Ile Gln Lys Glu Leu Cys Leu His                  530 535 540  Ala Ala Gln Gly Leu Val Gln Leu Lys Ala Cys Thr Tyr Lys Phe                  545 550 555  Leu Lys Met Cys Leu Ser Ala Asn Gly Glu His Pro Ser Leu Val                  560 565 570  Ser Cys Asn Pro Ser Asp Pro Leu Gln Lys Trp Lys Pro Leu Ile                  575 580 585  Met Tyr Thr Cys His Gly Leu Gly Gly Asn Gln Tyr Phe Glu Tyr                  590 595 600  Ser Ala Gln His Gly His Lys Thr Val Val Thr Gly Glu Gln Ile                  605 610 615  Trp Glu Ile Gln Lys Asp Gln Leu Leu Tyr Asn Pro Ile Leu Ser                  620 625 630  Gln Asn Asp              <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> sequence is synthesized <220> <221> misc_RNA <223> sequence is synthesized <400> 15  gaccatccgc agtgtatta 19 <210> 16 <211> 19 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> misc_RNA <223> Sequence is synthesized <400> 16  atacagatat gttcggtga 19 <210> 17 <211> 19 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> misc_RNA <223> sequence is synthesized <400> 17  ccatagatct gaacacgtt 19 <210> 18 <211> 19 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> misc_RNA <223> sequence is synthesized <400> 18  gcaaggatat tatacagca 19 <210> 19 <211> 19 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> misc_RNA <222> 2 <223> sequence is synthesized <220> <221> misc_RNA <222> 18 <223> sequence is synthesized <400> 19  guaccagaca cgcggcaua 19 <210> 20 <211> 19 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> misc_RNA <222> 10 <223> sequence is synthesized <220> <221> misc_RNA <222> 13 <223> sequence is synthesized <220> <221> misc_RNA <222> 15 <223> sequence is synthesized <400> 20  accgagaggu cauguacaa 19 <210> 21 <211> 19 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> misc_RNA <223> sequence is synthesized <400> 21  ggacaaagtt taccaaatg 19 <210> 22 <211> 59 <212> PRT <213> Homo sapiens <400> 22  Lys Arg Ser Ser Pro Ser Glu Gly Pro Cys Thr Thr Thr Arg Asn    1 5 10 15  Thr Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Ser Gly Thr                   20 25 30  Lys His Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr Ser                   35 40 45  Ser Pro Gly Thr Pro Ala Ser Pro Cys Ser Leu Ser Gly Ile                   50 55 <210> 23 <211> 30 <212> PRT <213> Homo sapiens <400> 23  Lys Arg Ser Ser Pro Ser Glu Gly Pro Cys Thr Thr Thr Arg Asn    1 5 10 15  Thr Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Ser Gly Ile                   20 25 30 <210> 24 <211> 28 <212> PRT <213> Homo sapiens <400> 24  Trp Glu His Ser Pro Leu Gly Glu Pro Cys Thr Thr Thr Arg Asn    1 5 10 15  Thr Ala Cys Gln Cys Lys Pro Gly Thr Phe Arg Glu Ser                   20 25 <210> 25 <211> 28 <212> PRT <213> Mus musculus <400> 25  Pro Glu Glu Ser Pro Ser Arg Gly Arg Cys Asn Ile Thr Thr Asn    1 5 10 15  Thr Val Cys Arg Cys Lys Pro Gly Thr Phe Glu Thr Ala                   20 25 <210> 26 <211> 28 <212> PRT <213> Homo sapiens <400> 26  Pro Pro Gly Glu Arg Lys Ala Arg Asn Cys Thr Arg Thr Gln Asn    1 5 10 15  Thr Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys Gly Ser                   20 25 <210> 27 <211> 28 <212> PRT <213> Homo sapiens <400> 27  Pro Gly Gln Asp Thr Asp Cys Arg Ser Cys Gln Glu Lys Gln Asn    1 5 10 15  Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Asp Ser                   20 25 <210> 28 <211> 40 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 28  acgctgctca ggaccattcg ctgcgtgtta aaccgcaccc 40 <210> 29 <211> 19 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 29  ctggtagcgg tcacataat 19  

Claims (24)

A mammalian cancer tissue or cancer cell sample is examined in vitro by immunohistochemistry to detect expression of GalNac-T14, wherein the immunohistochemistry is amino acid 1-552 or amino acid 39- of FIG. 4A. Using an anti-GalNac-T14 antibody that binds to a human GalNac-T14 polypeptide comprising 552, wherein expression of the GalNac-T14 indicates that the tissue or cell sample is sensitive to apoptosis-inducing activity of the Apo2L / TRAIL polypeptide. A method of predicting the sensitivity of a mammalian cancer tissue or cancer cell sample to Apo2L / TRAIL polypeptide as predicted. The method of claim 1, wherein the anti-GalNac-T14 antibody is a monoclonal antibody that binds to human GalNac-T14 comprising amino acids 1-552 of FIG. 4A. The method of claim 1, further comprising examining in vitro expression of a DR4, DR5, DcR1 or DcR2 receptor in said tissue or cell sample. The method of claim 1, wherein the cancer cell is pancreatic cancer, lymphoma, non-small cell lung cancer, colon cancer, colorectal cancer, melanoma or chondrosarcoma cell or tissue. Irradiating a mammalian cancer tissue or cancer cell sample in vitro with immunohistochemistry to detect expression of GalNac-T14, and Following detection of the expression of GalNac-T14, exposing the tissue or cell sample to an effective amount of Apo2L / TRAIL polypeptide in vitro Wherein the immunohistochemistry is performed using an anti-GalNac-T14 antibody that binds to a human GalNac-T14 polypeptide comprising amino acids 1-552 or amino acids 39-552 of FIG. 4A, wherein A method of inducing apoptosis in mammalian cancer tissue or cancer cell sample, wherein the expression predicts that the tissue or cell sample is sensitive to apoptosis-inducing activity of the Apo2L / TRAIL polypeptide. The method of claim 5, further comprising examining in vitro expression of DR4, DR5, DcR1 or DcR2 receptors in said tissue or cell sample. The method of claim 5, wherein the cancer cells are pancreatic cancer, lymphoma, non-small cell lung cancer, colon cancer, colorectal cancer, melanoma or chondrosarcoma cells or tissue. The method of claim 5, wherein said cells are exposed to an effective amount of Apo2L / TRAIL polypeptide comprising amino acids 114-281 of FIG. 1 (SEQ ID NO: 1). The method of claim 5, wherein said cells are also exposed to chemotherapeutic agents or radiation therapy. The method of claim 5, wherein said cells are also exposed to cytokines, cytotoxic agents, or growth inhibitory agents. The method of claim 5, wherein said Apo2L / TRAIL polypeptide is linked to a polyethylene glycol molecule. The method of claim 5, wherein the Apo2L / TRAIL polypeptide comprises amino acids 41-281 of FIG. 1 (SEQ ID NO: 1) or a biologically active fragment thereof. The method of claim 5, wherein said cancer cell is a non-small cell lung cancer cell. The method of claim 12, wherein the Apo2L / TRAIL polypeptide consists of amino acids 114-281 of FIG. 1 (SEQ ID NO: 1). An antibody or polynucleotide for detecting expression of GalNac-T14 protein or mRNA in a mammalian cancer tissue or cancer cell sample, wherein expression of the GalNac-T14 causes the tissue or cell sample to undergo apoptosis-inducing of Apo2L / TRAIL A kit or product for predicting sensitivity to Apo2L / TRAIL of a mammalian tissue or cell sample, which predicts sensitivity to activity. The kit or article of claim 15, wherein the expression of GalNac-T14 is investigated by detecting the expression of GalNac-T14 mRNA. The kit or article of claim 15, wherein the expression of GalNac-T14 is examined by immunohistochemistry. The kit or article of claim 15, wherein said detecting further comprises examining expression of a DR4, DR5, DcR1 or DcR2 receptor in said tissue or cell sample. The kit or product according to claim 15, wherein the cancer cells are pancreatic cancer, lymphoma, non-small cell lung cancer, colon cancer, colorectal cancer, melanoma or chondrosarcoma cells or tissue. Instructions for using a GalNac-T14 antibody to assess the presence of a GalNac-T14 in a container, a label on the container, a composition contained within the container, and one or more types of mammalian cells, wherein the composition Mammalian tissue comprising a primary antibody that binds to GalNac-T14, wherein the label on the container indicates that the composition can be used to assess the presence of GalNac-T14 in one or more types of mammalian cells Or a kit or product for predicting whether a cell is sensitive to induction of apoptosis by Apo2L / TRAIL polypeptide. The kit or article of claim 20, wherein the antibody is a GalNac-T14 monoclonal antibody. Instructions for using the polynucleotide to assess the presence of a container, a label on the container, a composition contained within the container, and GalNac-T14 RNA or DNA in one or more types of mammalian cells, wherein the The composition comprises a polynucleotide that hybridizes to a complement of GalNac-T14 polynucleotides under at least moderate stringency conditions, and the label on the container allows the composition to assess the presence of GalNac-T14 in one or more types of mammalian cells. A kit for predicting whether a mammalian tissue or cell will be sensitive to induction of apoptosis by Apo2L / TRAIL polypeptide, indicating that it may be used. delete delete

Images